Human monoclonal antibodies that bind insulin-like growth factor (IGF) I and II

ABSTRACT

Disclosed herein are human monoclonal antibodies that specifically bind both IGF-I and IGF-II with picomolar affinity and potently inhibit the IGF-IR signal transduction function. These antibodies are active in both an IgG and a scFv format. Bispecific forms of these antibodies are also disclosed. Nucleic acids encoding these antibodies, vectors including these nucleic acids, and host cells transformed with these vectors are also disclosed herein. Also disclosed are pharmaceutical compositions including these antibodies. Methods are provided for treating a subject with cancer and for inhibiting phosphorylation of the insulin-like growth factor-I receptor. Methods are also provided for diagnosing cancer.

CROSS REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage of PCT Application No.PCT/US2012/033128, filed Apr. 11, 2012, which was published in Englishunder PCT Article 21(2), which claims the benefit of U.S. ProvisionalApplication No. 61/474,664, filed Apr. 12, 2011, and the benefit of U.S.Provisional Application No. 61/548,164, filed Oct. 17, 2011, which isare both incorporated herein by reference.

FIELD

This application relates to the field of antibodies, specifically tohuman antibodies that specifically bind insulin-like growth factor(IGF)-I and IGF-II, and their use.

BACKGROUND

The insulin-like growth factor-I receptor (IGF-IR) and its ligands(IGF-I and IGF-II) have been implicated in a variety of physiologicprocesses and in pathologic conditions such as cancer (see, for example,Pollack et al., Nat Rev Cancer, 2008. 8(12): p. 915-28). Although therole of the IGF system in cancer has been recognized many years ago, itis not until recently that the system's components have been targetedand shown to affect cell transformation, proliferation, survival,motility, and migration in tissue cultures and in mouse models of cancer(see, for example, Wang et al., Curr Cancer Drug Targets, 2002. 2(3): p.191-207). The IGF-mediated signaling is initiated by binding of eitherIGF-I or IGF-II to their receptor (IGF-IR). Then phosphorylated IGF-IRrecruits adaptor proteins, such as insulin receptor substrate (IRS) 1,IRS2 and Src-Homology Collagen (SHC) (Feng et al., Curr Opin Drug DiscovDevel, 2008. 11(2): p. 178-85) Mechanistic studies have shown thatligand mediates the stimulation of IGF-IR, inducing receptor clusteringand autophosphorylation followed by transphosphorylation of the βsubunits (Hernandez-Sanchez et al., J Biol Chem, 1995. 270(49): p.29176-81). The phosphorylation of IRS1 regulates the activity ofphosphoinositide 3-kinase and protein kinase B (also known as Akt) andtriggers transcription factors which control the expression of manygenes that are important for cell proliferation and growth (Foulstone etal., J Pathol, 2005. 205(2): p. 145-53). Numerous studies demonstratedthat IGF-IR is expressed in a broad panel of tumors, suggesting thatinhibition of IGF-IR signaling may have both proapoptotic andantiproliferative consequences (Zha et al., Mol Cancer Ther., 2009.8(8): p. 2110-21). Thus, it has been proposed that modulation of theactivity of the IGF system could add to the arsenal of anticancertherapeutic approaches (Feng et al., Mol. Cancer. Ther., 2006. 5(1): p.114-20). A number of epidemiologic studies have shown consistently thathigh circulating levels of a potent mitogen, insulin-like growth factor(IGF)-I, are associated with increased risk for several common cancers,including those of the breast, prostate, lung, and colorectum. The levelof IGF-binding protein (IGFBP)-3, a major IGF-I-binding protein in serumthat, in most situations, suppresses the mitogenic action of IGF-I, isinversely associated with the risk of these cancers.

There is increasing epidemiological evidence to link elevated plasmaIGF-I level with prostate, breast, and colon cancer risk. Breast cancertissues from patients exhibit higher IGFR1 expression than adjacentnormal tissue, suggesting a link between IGFR1 and breast epithelialcell transformation. It has been reported that the transformationcapacity of tumor cells is attenuated when IGFR1 is inhibited using anantisense strategy, neutralizing antibody (anti-IR3 or anti-IGF-I) ordominant negative truncation of the receptor (see Hailey, J. et al,Molecular Cancer Therapeutics 1: 1349-1353, 2002; Maloney E. K., et al,Cancer Res. 63: 5073-5083, 2003; Burtrum D., et al, Cancer Res., 63:8912-8921, 2003; u et al., J. Biol. Chem. 279: 2856-2865, 2004; Miyamotoet al., Clin. Cancer Res. 11: 3494-3502, 2005; Goya et al., CancerResearch 64: 6252-6258, 2004). However, a need exists in the art forimproved multi-target therapies to treat neoplastic disease andmetastatic cancers.

SUMMARY

Disclosed herein are human monoclonal antibodies that specifically bindboth IGF-I and IGF-II with picomolar affinity and potently inhibit theIGF-IR signal transduction function. These antibodies are active in bothan IgG and a scFv format.

In some embodiments, the monoclonal human antibodies specifically bindinsulin-like growth factor II (IGF-II) with an equilibrium dissociationconstant (K_(d)) of 200 pM or less and specifically bind IGF-I with anequilibrium dissociation constant (K_(d)) of 200 pM or less, andinhibits phosphorylation of the insulin-like growth factor receptor. Inone specific non-limiting example, the human monoclonal antibody bindsIGF-I with an equilibrium constant (K_(d)) of 200 pM and IGF-II with anequilibrium constant (K_(d)) of 60 pM. In additional embodiments, themonoclonal human antibody inhibits the motility of breast cancer cellsin vitro. Bispecific forms of these antibodies are also disclosed.

In further embodiments, the heavy chain variable region of the antibodyincludes the amino acid sequence set forth as amino acids 26-33 of SEQID NO: 7, amino acids 51-58 of SEQ ID NO: 7, and/or amino acids 97-109of SEQ ID NO: 7. In yet other embodiments, the light chain variableregion of the antibody includes the amino acid sequence set forth asamino acids 27-32 of SEQ ID NO: 8, amino acids 50-52 of SEQ ID NO: 8and/or amino acids 89-97 of SEQ ID NO: 8. The antibodies can include theframework regions included in one or more of SEQ ID NOs: 7-10.

Nucleic acids encoding these antibodies, vectors including these nucleicacids, and host cells transformed with these vectors are also disclosedherein. Also disclosed are pharmaceutical compositions comprising theseantibodies, nucleic acids and vectors.

In some embodiments, methods are provided for treating a subject withcancer. These methods include administering to the subject atherapeutically effective amount of the monoclonal antibodies, nucleicacids and/or vectors, thereby treating the subject.

Methods are also disclosed for diagnosing cancer in a subject. Thesemethods include contacting a sample from the subject with an isolatedmonoclonal antibody that specifically binds both IGF-I and IGF-II withpicomolar affinity, and detecting binding of the isolated monoclonalantibody to the sample. An increase in the binding of the antibody tothe sample as compared to a control indicates that the subject hascancer.

In additional embodiments, methods are provided for inhibitingphosphorylation of the insulin-like growth factor-I receptor. Thesemethods include contacting a cell with an effective amount of theisolated monoclonal antibody that specifically binds both IGF-I andIGF-II with picomolar affinity, thereby inhibiting the phosphorylationof the insulin-like growth factor receptor.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph illustrating binding of Fab m705,6,8 to IGF-I, -IIas measured by ELISA. Fabs were added to wells coated with IGF-I (black)and IGF-II (white). Bound Fabs were detected with a mouse anti-Flag tagantibody and measured as optical densities at 450 nm.

FIG. 2 is a schematic diagram of an affinity maturation scheme. Themutagenesis yeast library was constructed from parental m708.2. Thelibrary was subjected to screen on IGF-I conjugated magnetic beads onceand sorted three times by FACS for binding to IGF-I. The sortedpopulation was mutated by error-prone PCR of the entire gene to yield anew sub-library. The process of sorting and mutagenesis was thencyclically repeated. The rounds of affinity maturation are named for thenumber of mutagenesis cycles, thus sorting of the parental library isround 1, followed by round 2 and 3. The highest affinity clone presentin the final round of maturation was identified and sequence analysis.

FIG. 3A to 3H is a set of plots showing FACS selection for affinitymaturation. Yeast libraries were labeled with mouse anti-c-myc antibodyfollowed by goat anti-mouse dye as well as biotinylated IGF-I followedby streptavidin-dye (a, b, c). During three FACS selections of round 1,yeast cells were stained with concentrations of IGF-I at 3 nM, 1 nM and0.3 nM, respectively (d, e). During two FACS selections of round 2,yeast cells were stained with concentrations of IGF-I at 3 nM, and 0.3nM, respectively (f, g, h). During three FACS selections of round 3,yeast cells were stained with concentrations of IGF-I at 1 nM, 0.5 nMand 0.1 nM. The 0.1-0.3% cells were selected from sort gates.

FIGS. 4A and 4B is a set of graphs showing a binding comparison of IgGsof m708.2 and m708.5. IgGs of m708.2 and m708.5 with serial dilutionswere added to wells coated with IGF-I (a) and IGF-II (b). Bound IgGswere detected with a HRP conjugated anti-human Fc antibody and measuredas optical densities at 450 nm.

FIG. 5A to 5D is a set of graphs showing inhibition of IGF-I and IGF-IIbinding to MCF-7 cells by m708.5 (a-b). M708.5 scFv and control 3A2ascFv were pre-incubated with biotinylated IGF-I and biotinylated IGF-IIfor 20 min at room temperature. Then mixtures were incubated with MCF-7cells for 30 min on ice (c-d). M708.5 IgG and control m102.4 IgG werepre-incubated with biotinylated IGF-I and biotinylated IGF-II for 20 minat room temperature. The mixtures then were incubated with MCF-7 cellsfor 30 min on ice. After the staining of R-phycoerythrin conjugatedStreptavidin for 30 min on ice, cells were detected by flow cytometry.

FIGS. 6A and 6B is a set of digital images showing phosphorylationinhibition of IGF-IR by m708.2 and m708.5 in MCF-7 cells. MCF-7 cellswere starved in serum free medium for 5 h first, followed by addition oftreatment medium with 1 nM IGF-I (a) or 5 nM IGF-II (b) with theindicated concentrations of IgG708.2. Thirty minutes later cells werechilled and lysed. IGF-IR was immunoprecipitated, the phosphorylatedIGF-IR was detected with an phosphotyrosine specific antibody. The totalamount of IGF-IR was detected by the same polyclonal antibody used forthe immunoprecipitation.

FIGS. 7A and 7B are a sets of digital images. (a) Phosphorylationinhibition of IR by m708.2, m708.5 and m610 in MCF-7 cells. MCF-7 cellswere starved and treated with 5 nM IGF-II with indicated concentrationsof IgGs. The phosphorylated IR was detected with a phospho-tyrosinespecific antibody. The total amount of IR was detected. (b) ELISA ofm708.5 to human insulin. IgGs of m708.5 (10 nM) were added to wellscoated with IGF-I, IGF-II, human insulin and irrelevant antigens. BoundIgGs were detected with a HRP conjugated anti-human Fc antibody andmeasured as optical densities at 450 nm.

FIG. 8 is a graph of growth inhibition of MCF-7 cells by m708.2 andm708.5. MCF-7 cells were incubated in complete medium overnight.Different concentrations of IgG m708.2 and m708.5 were pre-incubatedwith added IGF-I (2.5 nM) and IGF-II (2.5 nM) for 15 min. The media ofMCF-7 cells were replaced by the mixture of IgG and ligands immediately.Cells were allowed to grow for 3 days, and MTS substrate was added todetect viable cells. The reaction was monitored by absorbance at 450 nM.Positive control was cells in serum free medium with IGF ligands. Blankcontrol was cells in serum free medium without any IGF ligands.

FIG. 9 is an alignment showing a comparison of the m708.5 heavy chain(SEQ ID NO: 7) with m708.2 heavy chain (SEQ ID NO: 9) and a comparisonof the m708.5 light chain (SEQ ID NO: 8) with the m708.2 light chain(SEQ ID NO: 10). The positions (amino acid numbers) are shown above thesequences. CDRs are highlighted; framework regions are not highlighted.For m708.5, H-CDR1 is amino acids 26-33 of SEQ ID NO: 7, H-CDR2 is aminoacids 51-58 of SEQ ID NO: 7, and H-CDR3 is amino acids 97-109 of SEQ IDNO: 7, L-CDR1 is amino acids 27-32 of SEQ ID NO: 8, L-CDR2 is aminoacids 50-52 of SEQ ID NO: 8, and L-CDR3 is amino acids 89-97 of SEQ IDNO: 8. Thus, amino acids 1-25, amino acids 34-50, amino acids 59-96 andamino acids 110-120 of SEQ ID NO: 7 and SEQ ID NO: 9 are heavy chainframework regions. Amino acids 1-26, amino acids 33-49, amino acids53-88 and amino acids 98-108 of SEQ ID NO: 8 and SEQ ID NO: 10 are lightchain framework regions.

FIG. 10 is an alignment showing a comparison of the m708.5 heavy chainwith the m708.6 heavy chain and the m708.7 heavy chain and a comparisonof the m7085. light chain with the m708.6 light chain. The heavy chainamino acid sequences are all encompassed by the consensus sequence aminoacid set forth as SEQ ID NO: 7. The light chain amino acid sequences areall encompassed by the consensus amino acid sequence set forth as SEQ IDNO: 8.

FIG. 11 is a schematic representation of the bispecific antibody, m67.This bispecific antibody was generated by fusing scFv m610.27 and scFvm708.5 to the N terminus of the heavy and light chain constant regionsof a human IgG1, respectively, using linkers of G₄5 (SEQ ID NO: 25)triplicates.

FIGS. 12A and 12B are line graphs showing competition of m610 withm708.5 in binding to IGF-II. (FIG. 12A) IGF-II was directly coated onthe ELISA plate. Bound scFv m610 was detected by HRP-conjugatedanti-Flag antibody in the presence of antibody competitors (IgG1 m708.5or IgG1 m102.4. (FIG. 12B) IgG1 m610.27 was coated on the ELISA plateand IGF-II was captured by coated IgG1 m610.27. Bound scFv m708.5 or VHm630.3 was detected by HRP-conjugated anti-Flag tag antibody.

FIGS. 13A and 13B are graphs of results of size-exclusion chromatographyanalysis of antibodies in the presence of IGFs. (FIG. 13A) IgG1 m708.5or the mixture of IgG1 m610 and IgG1 m708.5 plus IGF-II was analyzed bySuperdex G75 column. (FIG. 13B) m67, the mixture of m67 and IGF-II, orthe mixture of m67/IGF-II plus IGF-I was analyzed by Superdex G200column.

FIGS. 14A and 14B are graphs showing stabilization of m67 and m708.5 inHuman Sera. Bispecific antibody (BsAb) m67 (FIG. 14A) and IgG1 m708.5(FIG. 14B) were incubated with equal volume of human sera at 37° C. for9 days and then tested to bind to IGF-I and IGF-II by ELISA.

FIGS. 15A and 15B are graphs showing binding Inhibition of IGF-I (FIG.15A) and IGF-II (FIG. 15B) on MCF7 Cells. MCF-7 cells were incubatedwith 5 nM biotinylated IGF-I or 1 nM biotinylated IGF-II in the absenceor presence of antibodies. Bound biotinylated IGF-I or IGF-II wasdetected by streptavidin-PE. Blank cells incubated with streptavidin-PEconjugate only are in grey. Cells incubated with IGF-I or IGF-II onlyare shown by a solid line. Those for IGF-I or IGF-II with antibodies areshown with a dashed line.

FIG. 16 is a set of graphs showing binding of antibodies to BJAB Cellsin the presence of IGF-II. Bound biotinylated IGF-I or IGF-II wasdetected by Streptavidin-PE. Blank cells incubated with Streptavidin-PEconjugate are in grey. Cells incubated with IGF-II only are indicated bya solid line. Those for IGF-II with antibodies are shown by a dashedline.

FIG. 17 is a set of graphs showing biding of antibodies to U937 Cells inthe presence of IGF-II. Bound antibodies were detected byFITC-conjugated goat F(ab′)2 anti-human Fc IgG antibody. The black(filled in) histograms are the blank cells incubated with the secondaryantibody only. The histograms for cells incubated with antibody alone(hatched area), the mixture of antibody and IGF-II (dashed line), andthe mixture of the antibody, IGF-II and Cytochalasin D (solid line) areshown.

FIGS. 18A and 18B are a set of digital images showing inhibition ofIGF1R and IR phosphorylation. MCF-7 cells were starved in serum freemedium for 5 hours first, followed by addition of treatment medium with1 nM IGF-I or 5 nM IGF-II with the indicated concentrations ofantibodies. Thirty minutes later cells were chilled and lysed. (FIG.18A) IGF1R was immunoprecipitated and the phosphorylated IGF1R wasdetected with a phosphor-tyrosine specific antibody. The total amount ofIGF1R were detected by the same polyclonal antibody used for theimmunoprecipitation. (FIG. 18B) IR was immunoprecipitated and detectedas following above methods.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing was submitted as an ASCII textfile (4239-86603-03_Sequence_Listing.txt), created on Dec. 3, 2014, 20.8KB, which is incorporated by reference herein.

SEQ ID NO: 1 is an exemplary amino acid sequence of a human insulinchain A.

SEQ ID NO: 2 is an exemplary amino acid sequence of a human insulinchain B.

SEQ ID NO: 3 is an exemplary amino acid sequence of an IGF-I precursor.

SEQ ID NO: 4 is an exemplary amino acid sequence of a mature IGF-I.

SEQ ID NO: 5 is an exemplary amino acid sequence of an IGF-II precursor.

SEQ ID NO: 6 is an exemplary amino acid sequence of a mature IGF-II.

SEQ ID NO: 7 is the amino acid sequence of a consensus sequence, whichencompasses human monoclonal antibody clone m708.5, m708.6 and m708.7heavy chain.

SEQ ID NO: 8 is the amino acid sequence of a consensus sequence, whichencompasses human monoclonal antibody clone m708.5, m708.6 and m708.7light chain.

SEQ ID NO: 9 is the amino acid sequence of human monoclonal antibodyclone m708.2 heavy chain.

SEQ ID NO: 10 is the amino acid sequence of human monoclonal antibodyclone m708.2 light chain.

SEQ ID NO: 11 is a nucleic acid sequence encoding human monoclonalantibody clone m708.5 heavy chain.

SEQ ID NO: 12 is a nucleic acid sequence encoding human monoclonalantibody clone m708.5 light chain.

SEQ ID NO: 13 is a nucleic acid sequence encoding human monoclonalantibody clone m708.2 heavy chain.

SEQ ID NO: 14 is a nucleic acid sequence encoding human monoclonalantibody clone m708.2 light chain.

SEQ ID NOs: 15-18 are the nucleic acid sequences of primers.

SEQ ID NO: 19 is the nucleic acid sequence encoding human monoclonalantibody clone m708.6 heavy chain.

SEQ ID NO: 20 is the nucleic acid sequence encoding human monoclonalantibody clone m708.6 light chain.

SEQ ID NO: 21 is the nucleic acid sequence encoding human monoclonalantibody clone m708.7 heavy chain.

SEQ ID NO: 22 is the nucleic acid sequence encoding human monoclonalantibody clone m708.7 light chain.

SEQ ID NO: 23 is the amino acid sequence of the m610.27 heavy chain.

SEQ ID NO: 24 is the amino acid sequence of the m610.27 light chain.

SEQ ID NO: 25 is a nucleic acid sequence encoding the m610.27 heavychain.

SEQ ID NO: 26 is a nucleic acid sequence encoding the m610.27 lightchain.

DETAILED DESCRIPTION

Disclosed herein are human monoclonal antibodies that specifically bindboth IGF-I and IGF-II and inhibit the IGF-IR signal transductionfunction. Thus, these antibodies inhibit phosphorylation of theinsulin-like growth factor receptor. These monoclonal human antibodiescan inhibit the motility of breast cancer cells in vitro. Additionalcompositions and methods are described below.

I. Abbreviations

-   -   BSA: bovine serum albumin    -   CDR: complementarity determining region    -   dsFv: disulfide stabilized fragment of a variable region    -   DMEM: Dulbecco's modified eagle medium    -   ELISA: enzyme-linked immunosorbent assay    -   EM: effector molecule    -   ERK: extra-cellular signal response kinase    -   FACS: fluorescence activated cell sorting    -   FBS: fetal bovine serum    -   FITC: fluoroscein isothiocyanate    -   HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   IGF-I: insulin-like growth factor I    -   IGF-IR: insulin-like growth factor I receptor    -   IGF-II: insulin-like growth factor II    -   IGFBP: insulin-like growth factor binding proteins    -   IGFBP-rP: IGFBP-related proteins    -   IPTG: isopropyl-beta-D-thiogalactopyranoside    -   HCDR: heavy chain complementarity determining region    -   HAMA: human anti-murine antibody    -   HAT: hypoxanthine aminopterin thymidine    -   IL-6: interleukin-6    -   Ig: immunoglobulin    -   IR: insulin receptor    -   IRR: insulin receptor-related receptor    -   kDa: kilodaltons    -   LCDR: light chain complementarity determining region    -   MAb: monoclonal antibody    -   MAPK: mitogen-activated protein kinase    -   MMP: matrix-metalloproteinase    -   PBS: phosphate buffered saline    -   scFv: single chain fragment of a variable region    -   SDR: specificity determining residues    -   SDS-PAGE: sodium dodecyl(lauryl) sulfate-polyacrylamide gel        electrophoreses    -   RIA: radioimmunoassay    -   V_(H): variable region of a heavy chain    -   V_(L): variable region of a light chain

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Administration: The introduction of a composition into a subject by achosen route. For example, if the chosen route is intravenous, thecomposition is administered by introducing the composition into a veinof the subject.

Amplification: Of a nucleic acid molecule (such as, a DNA or RNAmolecule) refers to use of a technique that increases the number ofcopies of a nucleic acid molecule in a specimen. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of amplification maybe characterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen, such as insulin-like growth factor(IGF)-I, IGF-II, or a fragment thereof. In vivo, antibodies are composedof a heavy and a light chain, each of which has a variable region,termed the variable heavy (V_(H)) region and the variable light (V_(L))region. Together, the V_(H) region and the V_(L) region are responsiblefor binding the antigen recognized by the antibody, such that theantibody specifically binds the antigen.

This includes intact immunoglobulins and the variants and portions ofthem well known in the art, such as Fab′ fragments, F(ab)′₂ fragments,single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies (for example, humanized murineantibodies), heteroconjugate antibodies (such as, bispecificantibodies). See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H.Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDRs inthree-dimensional space.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody with a defined variable domain,such as a monoclonal antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds IGF-II.

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. A “humanized” immunoglobulin is animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (for example a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one embodiment, all the CDRs are from thedonor immunoglobulin in a humanized immunoglobulin. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. A “humanized antibody” is an antibody comprising a humanizedlight chain and a humanized heavy chain immunoglobulin. A humanizedantibody binds to the same antigen as the donor antibody that providesthe CDRs. The acceptor framework of a humanized immunoglobulin orantibody may have a limited number of substitutions by amino acids takenfrom the donor framework. Humanized or other monoclonal antibodies canhave additional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Humanized immunoglobulins can be constructed by means ofgenetic engineering (see for example, U.S. Pat. No. 5,585,089).

Binding affinity: Affinity of an antibody for an antigen, such as IGF-Iand IGF-II. In one embodiment, affinity is calculated by a modificationof the Scatchard method described by Frankel et al., Mol. Immunol.,16:101-106, 1979. In another embodiment, binding affinity is measured byan antigen/antibody dissociation rate.

In yet another embodiment, a high binding affinity is measured by acompetition radioimmunoassay. In several examples, a high bindingaffinity is at least a picomolar binding affinity. In other examples, ahigh binding affinity is 10 pm, 50 pM, 60 pM, 100 pM, 200 pM or 300 pMfor IGF-I and/or IGF-II. Generally, a K_(D) for an antibody with highbinding affinity is 1×10⁻¹⁰ M or less. In other embodiments, a highbinding affinity is a K_(D) of about 1.5×10⁻¹⁰, about 2.0×10⁻¹⁰, about2.5×10⁻¹⁰, about 3.0×10⁻¹⁰, about 3.5×10⁻¹⁰, about 4.0×10⁻¹⁰, about4.5×10⁻¹⁰, or about 5.0×10⁻¹⁰ M or less. In further embodiments, a highbinding affinity is a K_(D) of about 1.5×10⁻¹¹, about 2.0×10⁻¹¹, about2.5×10⁻¹¹, about 3.0×10⁻¹¹, about 3.5×10⁻¹¹, about 4.0×10⁻¹¹, about4.5×10⁻¹¹, or about 5.0×10⁻¹¹ M or less. In additional embodiments, ahigh binding affinity is a K_(D) of about 1.5×10⁻¹², about 2.0×10⁻¹²,about 2.5×10⁻¹², about 3.0×10⁻¹², about 3.5×10⁻¹², about 4.0×10⁻¹²,about 4.5×10⁻¹², or about 5.0×10⁻¹² M or less.

Bi-specific antibody: A recombinant molecule composed of two differentantigen binding moieties and consequently binds to two differentantigenic epitopes. Bi-specific antibodies include chemically orgenetically linked molecules of two antigen-binding moieties. Theantigen binding moieties can be linked using a linker. The antigenbinding moieties can be monoclonal antibodies, antigen-binding fragments(e.g., Fab, scFv), eAds, or combinations thereof. A bispecific antibodycan include one or more constant domains, but does not necessarilyinclude a constant domain.

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is an agent of use in treating alymphoma, leukemia, or another tumor. In one embodiment, achemotherapeutic agent is a radioactive compound. One of skill in theart can readily identify a chemotherapeutic agent of use (see forexample, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds): OncologyPocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995;Fischer, D. S., Knobf, M. F., Durivage, H. J. (eds): The CancerChemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).Combination chemotherapy is the administration of more than one agent totreat cancer. One example is the administration of an antibody thatbinds IGF-II or a fragment thereof used in combination with aradioactive or chemical compound.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease the affinityof an antibody to IGF-I or IGF-II. For example, a human antibody thatspecifically binds IGF-I and IGF-II can include at most about 1, at mostabout 2, at most about 5, and most about 10, or at most about 15conservative substitutions and specifically bind the original IGF-I andIGF-II polypeptide with a similar affinity. The term conservativevariation also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid, provided that antibody specificallybinds IGF-I and IGF-II. Non-conservative substitutions are those thatreduce an activity or binding to IGF-I and/or IGF-II.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Complementarity Determining Region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,H-CDR2, H-CDR3, respectively. By definition, the CDRs of the light chainare bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 58(L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are bounded bythe residues at positions 31 and 35b (H-CDR1), 50 and 65 (H-CDR2), 95and 102 (H-CDR3), using the numbering convention delineated by Kabat etal., (1991) Sequences of Proteins of Immunological Interest, 5^(th)Edition, U.S. Department of Health and Human Services, Public HealthService, National Institutes of Health, Bethesda, Md. (NIH PublicationNo. 91-3242).

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds IGF-I and IGF-II will havea specific V_(H) region and the V_(L) region sequence, and thus specificCDR sequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

Contacting: Placement in direct physical association; includes both insolid and liquid form.

Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to thecells intended to be targeted, as opposed to the cells of the rest of anorganism. In one embodiment, in contrast, the term “toxicity” refers totoxicity of an immunotoxin to cells other than those that are the cellsintended to be targeted by the targeting moiety of the immunotoxin, andthe term “animal toxicity” refers to toxicity of the immunotoxin to ananimal by toxicity of the immunotoxin to cells other than those intendedto be targeted by the immunotoxin.

Degenerate variant: A polynucleotide encoding an IGF-I and/or an IGF-IIpolypeptide or an antibody that binds IGF-I and IGF-II that includes asequence that is degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included aslong as the amino acid sequence of the IGF-I and/or IGF-II polypeptideor antibody that binds IGF-I and IGF-II encoded by the nucleotidesequence is unchanged.

Effector molecule: The portion of a chimeric molecule that is intendedto have a desired effect on a cell to which the chimeric molecule istargeted. Effector molecule is also known as an effector moiety (EM),therapeutic agent, or diagnostic agent, or similar terms.

Therapeutic agents include such compounds as nucleic acids, proteins,peptides, amino acids or derivatives, glycoproteins, radioisotopes,lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeuticand diagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides. Alternatively, the molecule linkedto a targeting moiety, such as an anti-IGF-II antibody, may be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (such as anantisense nucleic acid), or another therapeutic moiety that can beshielded from direct exposure to the circulatory system. Means ofpreparing liposomes attached to antibodies are well known to those ofskill in the art. See, for example, U.S. Pat. No. 4,957,735; and Connoret al., Pharm. Ther. 28:341-365, 1985. Diagnostic agents or moietiesinclude radioisotopes and other detectable labels. Detectable labelsuseful for such purposes are also well known in the art, and includeradioactive isotopes such as ³²P, ¹²⁵I, and ¹³¹I, fluorophores,chemiluminescent agents, and enzymes.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide.

Expressed: Translation of a nucleic acid into a protein. Proteins may beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Framework Region: Amino acid sequences interposed between CDRs. Includesvariable light and variable heavy framework regions. The frameworkregions serve to hold the CDRs in an appropriate orientation for antigenbinding.

HAMA (Human anti-murine antibody) response: An immune response in ahuman subject to the variable and constant regions of a murine antibodythat has been administered to the patient. Repeated antibodyadministration may lead to an increased rate of clearance of theantibody from the patient's serum and may also elicit allergic reactionsin the patient.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody. The effector molecule can be a detectable label or animmunotoxin. Specific, non-limiting examples of toxins include, but arenot limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35,PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, ormodified toxins thereof, or other toxic agents that directly orindirectly inhibit cell growth or kill cells. For example, PE and DT arehighly toxic compounds that typically bring about death through livertoxicity. PE and DT, however, can be modified into a form for use as animmunotoxin by removing the native targeting component of the toxin(such as the domain Ia of PE and the B chain of DT) and replacing itwith a different targeting moiety, such as an antibody. A “chimericmolecule” is a targeting moiety, such as a ligand or an antibody,conjugated (coupled) to an effector molecule. The term “conjugated” or“linked” refers to making two polypeptides into one contiguouspolypeptide molecule. In one embodiment, an antibody is joined to aneffector molecule (EM). In another embodiment, an antibody joined to aneffector molecule is further joined to a lipid or other molecule to aprotein or peptide to increase its half-life in the body. The linkagecan be either by chemical or recombinant means. In one embodiment, thelinkage is chemical, wherein a reaction between the antibody moiety andthe effector molecule has produced a covalent bond formed between thetwo molecules to form one molecule. A peptide linker (short peptidesequence) can optionally be included between the antibody and theeffector molecule. Because immunoconjugates were originally preparedfrom two molecules with separate functionalities, such as an antibodyand an effector molecule, they are also sometimes referred to as“chimeric molecules.” The term “chimeric molecule,” as used herein,therefore refers to a targeting moiety, such as a ligand or an antibody,conjugated (coupled) to an effector molecule.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence, such as an N-terminal repeat, such that the peptidewill bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”)response, or a B cell response (e.g. antibody production) against theantigen from which the immunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide. In one specific non-limitingexample, an immunogenic polypeptide includes a region of IGF-I orIGF-II, or a fragment thereof, wherein the polypeptide that is expressedon the cell surface of a host cell that expresses the full-length IGF-IIpolypeptide.

Immunogenic composition: A composition comprising an IGF-I and/or IGF-IIpolypeptide that induces a measurable CTL response against cellsexpressing IGF-I and/or an IGF-II polypeptide, or induces a measurable Bcell response (such as production of antibodies) against an IGF-I and/oran IGF-II polypeptide. It further refers to isolated nucleic acidsencoding an IGF-I and/or an IGF-II polypeptide that can be used toexpress the polypeptide(s) (and thus be used to elicit an immuneresponse against this polypeptide). For in vitro use, an immunogeniccomposition may consist of the isolated protein or peptide epitope. Forin vivo use, the immunogenic composition will typically comprise theprotein or immunogenic peptide in pharmaceutically acceptable carriers,and/or other agents. Any particular peptide, such as an IGF-I or anIGF-II polypeptide, or nucleic acid encoding the polypeptide, can bereadily tested for its ability to induce a CTL or B cell response byart-recognized assays. Immunogenic compositions can include adjuvants,which are well known to one of skill in the art.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, pH) that are typical insidea living mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (i.e., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Immunotherapy: A method of evoking an immune response against cancercells based on their production of target antigens. Immunotherapy basedon cell-mediated immune responses involves generating a cell-mediatedresponse to cells that produce particular antigenic determinants, whileimmunotherapy based on humoral immune responses involves generatingspecific antibodies to cells that produce particular antigenicdeterminants.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as a tumor (for example, a cancer such as a leukemia or acarcinoma). “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop. As used herein, the term “ameliorating,”with reference to a disease or pathological condition, refers to anyobservable beneficial effect of the treatment. The beneficial effect canbe evidenced, for example, by a delayed onset of clinical symptoms ofthe disease in a susceptible subject, a reduction in severity of some orall clinical symptoms of the disease, a slower progression of thedisease, a reduction in the number of metastases, an improvement in theoverall health or well-being of the subject, or by other parameters wellknown in the art that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

Isolated: An “isolated” biological component (such as a nucleic acid,protein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In one example, a “labeled antibody” refers to incorporationof another molecule in the antibody. For example, the label is adetectable marker, such as the incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (such as ³⁵S or ¹³¹I), fluorescent labels(such as fluoroscein isothiocyanate (FITC), rhodamine, lanthanidephosphors), enzymatic labels (such as horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (such as a leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags), or magnetic agents, such as gadolinium chelates. In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

Ligand: Any molecule which specifically binds an IGF-I and/or an IGF-IIprotein and includes, inter alia, antibodies that specifically bind anIGF-I and/or IGF-II protein. In alternative embodiments, the ligand is aprotein or a small molecule (one with a molecular weight less than 6kiloDaltons).

Linker peptide: A peptide within an antibody binding fragment (such asan Fv fragment) which serves to indirectly bond the variable heavy chainto the variable light chain. “Linker” can also refer to a peptideserving to link a targeting moiety, such as a scFv, to an effectormolecule, such as a cytotoxin or a detectable label.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an scFv. In the specific context, the terms include reference tojoining a ligand, such as an antibody moiety, to an effector molecule(“EM”). The linkage can be either by chemical or recombinant means.“Chemical means” refers to a reaction between the antibody moiety andthe effector molecule such that there is a covalent bond formed betweenthe two molecules to form one molecule.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Major Histocompatibility Complex or MHC: Generic designation meant toencompass the histocompatibility antigen systems described in differentspecies, including the human leukocyte antigens (“HLA”). The term“motif” refers to the pattern of residues in a peptide of definedlength, usually about 8 to about 11 amino acids, which is recognized bya particular MHC allele. The peptide motifs are typically different foreach MHC allele and differ in the pattern of the highly conservedresidues and negative binding residues.

Neoplasia and Tumor: The process of abnormal and uncontrolled growth ofcells. Neoplasia is one example of a proliferative disorder. The productof neoplasia is a neoplasm (a tumor), which is an abnormal growth oftissue that results from excessive cell division. The amount of a tumorin an individual is the “tumor burden” which can be measured as thenumber, volume, or weight of the tumor. A tumor that does notmetastasize is referred to as “benign.” A tumor that invades thesurrounding tissue and/or can metastasize is referred to as “malignant.”Examples of hematological tumors include leukemias, including acuteleukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

In several examples a tumor is a sarcoma, leukemia, prostate cancer,lung cancer, breast cancer, lung cancer, colon cancer, stomach cancer,uterine cancer, cervical cancer, esophageal cancer, liver cancer,pancreatic cancer, kidney cancer, thyroid cancer, brain cancer, or anovarian cancer

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, such as a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (such as a promoter, origin ofreplication, ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used.

Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482, 1981, by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see for example, Current Protocols in MolecularBiology (Ausubel et al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, such as version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The BLASTN program (for nucleotidesequences) uses as defaults a word length (W) of 11, alignments (B) of50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.The BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989).

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Peptide: A chain of amino acids of between 3 and 30 amino acids inlength. In one embodiment, a peptide is from about 10 to about 25 aminoacids in length. In yet another embodiment, a peptide is from about 11to about 20 amino acids in length. In yet another embodiment, a peptideis about 12 amino acids in length.

An “IGF-II peptide” is a series of contiguous amino acid residues froman IGF-II protein. An “IGF-I peptide” is a series of contiguous aminoacid residues from an IGF-I protein. In one example, with respect toimmunogenic compositions comprising an IGF-I or an IGF-II peptide, theterm further refers to variations of these peptides in which there areconservative substitutions of amino acids, so long as the variations donot alter by more than about 20% (such as no more than about 1%, about5%, or about 10%) the ability of the peptide to produce a B cellresponse, or, when bound to a Major Histocompatibility Complex Class Imolecule, to activate cytotoxic T lymphocytes against cells expressingwild-type IGF-II protein. Induction of CTLs using synthetic peptides andCTL cytotoxicity assays are taught in, for example, U.S. Pat. No.5,662,907.

Peptide modifications: IGF-I and IGF-II polypeptides include syntheticembodiments of peptides described herein. In addition, analogs(non-peptide organic molecules), derivatives (chemically functionalizedpeptide molecules obtained starting with the disclosed peptidesequences) and variants (homologs) of these proteins can be utilized inthe methods described herein. Each polypeptide is comprised of asequence of amino acids, which may be either L- and/or D-amino acids,naturally occurring and otherwise.

Peptides may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the IGF-II peptides toselect and provide conformational constraints to the structure thatresult in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of a IGF-II polypeptide havingmeasurable or enhanced ability to generate an immune response. Forcomputer modeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs”, in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press, Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (such as a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA. A IGF-II polynucleotide is a nucleic acidencoding a IGF-II polypeptide.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (such as glycosylation orphosphorylation). In one embodiment, the polypeptide is IGF-IIpolypeptide. A “residue” refers to an amino acid or amino acid mimeticincorporated in a polypeptide by an amide bond or amide bond mimetic. Apolypeptide has an amino terminal (N-terminal) end and a carboxyterminal (C-terminal) end.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, and can be DNA oligonucleotides 15 nucleotides or more in length.Primers may be annealed to a complementary target DNA strand by nucleicacid hybridization to form a hybrid between the primer and the targetDNA strand, and then extended along the target DNA strand by a DNApolymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example, by the polymerase chain reaction(PCR) or other nucleic-acid amplification methods known in the art. Oneof skill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, for example, a primercomprising 20 consecutive nucleotides will anneal to a target with ahigher specificity than a corresponding primer of only 15 nucleotides.Thus, in order to obtain greater specificity, probes and primers may beselected that comprise 20, 25, 30, 35, 40, 50 or more consecutivenucleotides.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, forexample, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elementswhich can be located as much as several thousand base pairs from thestart site of transcription. Both constitutive and inducible promotersare included (see for example, Bitter et al., Methods in Enzymology153:516-544, 1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (such as the metallothioneinpromoter) or from mammalian viruses (such as the retrovirus longterminal repeat; the adenovirus late promoter; the vaccinia virus 7.5Kpromoter) may be used. Promoters produced by recombinant DNA orsynthetic techniques may also be used. A polynucleotide can be insertedinto an expression vector that contains a promoter sequence whichfacilitates the efficient transcription of the inserted genetic sequenceof the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific nucleic acid sequences thatallow phenotypic selection of the transformed cells.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Inone embodiment, a preparation is purified such that the protein orpeptide represents at least 50% of the total peptide or protein contentof the preparation.

The IGF-I and IGF-II polypeptides disclosed herein, or antibodies thatspecifically bind IGF-I and IGF-II, can be purified by any of the meansknown in the art. See for example Guide to Protein Purification, ed.Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; andScopes, Protein Purification: Principles and Practice, Springer Verlag,New York, 1982. Substantial purification denotes purification from otherproteins or cellular components. A substantially purified protein is atleast 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific,non-limiting example, a substantially purified protein is 90% free ofother proteins or cellular components.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques.

Recombinant toxins: Chimeric proteins in which a cell targeting moietyis fused to a toxin (Pastan et al., Science, 254:1173-1177, 1991). Ifthe cell targeting moiety is the Fv portion of an antibody, the moleculeis termed a recombinant immunotoxin (Chaudhary et al., Nature,339:394-397, 1989). The toxin moiety is genetically altered so that itcannot bind to the toxin receptor present on most normal cells.Recombinant immunotoxins selectively kill cells which are recognized bythe antigen binding domain. These recombinant toxins and immunotoxinscan be used to treat cancer, for example, cancers in which IGF-II isexpressed.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that exclude non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (such as GC versus ATcontent), and nucleic acid type (such as RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific, non-limiting example of progressively higher stringencyconditions is as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). One of skill in the art can readily determine variations onthese conditions (see Molecular Cloning: A Laboratory Manual, 2nd ed.,Vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, for example, high stringency conditions, or each ofthe conditions can be used, for example, for 10-15 minutes each, in theorder listed above, repeating any or all of the steps listed. However,as mentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a IGF-II polypeptide will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a IGF-II polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 90%,95%, 96%, 97%, 98% or 99% sequence identity counted over the full lengthalignment with the amino acid sequence of the antibody using the NCBIBlast 2.0, gapped blastp set to default parameters. For comparisons ofamino acid sequences of greater than about 30 amino acids, the Blast 2sequences function is employed using the default BLOSUM62 matrix set todefault parameters, (gap existence cost of 11, and a per residue gapcost of 1). When aligning short peptides (fewer than around 30 aminoacids), the alignment should be performed using the Blast 2 sequencesfunction, employing the PAM30 matrix set to default parameters (open gap9, extension gap 1 penalties). Proteins with even greater similarity tothe reference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus an IGF-II specific binding agent is an agent thatbinds substantially to a IGF-II polypeptide. In one embodiment, thespecific binding agent is a human monoclonal antibody that specificallybinds both IGF-I and IGF-II polypeptides. Thus an IGF-I and IGF-IIspecific binding agent is an agent that binds substantially to bothIGF-I and IGF-II polypeptides, but not to other unrelated polypeptides.In one embodiment, the specific binding agent is a human monoclonalantibody that specifically binds IGF-I and IGF-II polypeptides.

The term “specifically binds” refers, with respect to an antigen such asIGF-I and/or IGF-II, to the preferential association of an antibody orother ligand, in whole or part, with a cell or tissue bearing thatantigen and not to cells or tissues lacking that antigen. It is, ofcourse, recognized that a certain degree of non-specific interaction mayoccur between a molecule and a non-target cell or tissue. Nevertheless,specific binding may be distinguished as mediated through specificrecognition of the antigen. Although selectively reactive antibodiesbind antigen, they may do so with low affinity. On the other hand,specific binding results in a much stronger association between theantibody (or other ligand) and cells bearing the antigen than betweenthe bound antibody (or other ligand) and cells lacking the antigen.Specific binding typically results in greater than 2-fold, such asgreater than 5-fold, greater than 10-fold, or greater than 100-foldincrease in amount of bound antibody or other ligand (per unit time) toa cell or tissue bearing the IGF-I/IGF-II polypeptide as compared to acell or tissue lacking the polypeptide. Specific binding to a proteinunder such conditions requires an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsare appropriate for selecting antibodies or other ligands specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow & Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork (1988), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, CD8 Tcells are cytotoxic T lymphocytes. In another embodiment, a CD8 cell isa suppressor T cell.

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthof a tumor. In one embodiment, a therapeutically effective amount is theamount necessary to eliminate a tumor or reduce metastases. Whenadministered to a subject, a dosage will generally be used that willachieve target tissue concentrations (for example, in tumors) that hasbeen shown to achieve a desired in vitro effect.

Toxin: A molecule that is cytotoxic for a cell. Toxins include abrin,ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinumtoxin, saporin, restrictocin or gelonin, or modified toxins thereof. Forexample, PE and DT are highly toxic compounds that typically bring aboutdeath through liver toxicity. PE and DT, however, can be modified into aform for use as an immunotoxin by removing the native targetingcomponent of the toxin (such as domain Ia of PE or the B chain of DT)and replacing it with a different targeting moiety, such as an antibody.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Antibodies that Specifically Bind IGF-I and IGF-II

Disclosed herein are monoclonal antibodies that specifically bind bothIGF-I and IGF-II with a high affinity, such as picomolar affinity. Theseantibodies potently inhibit the IGF-1R signal transduction function.These antibodies are active in both an IgG and a scFv format, and can beincluded in bispecific antibodies.

The two ligands of the insulin like growth factor (IGF) system, IGF-Iand IGF-II, are single-chain polypeptides sharing 62% homology withproinsulin. Exemplary amino sequences of human insulin chain A, insulinchain B, IGF-I precursor, mature IGF-I, IGF-II precursor (also known as“long IGF-II”), and mature IGF-II are set forth in SEQ ID NOs:1, 2, 3,4, 5, and 6, respectively. The degree of homology between human andmouse IGF-I is 97%, while the degree of homology between human and mouseIGF-II is 91%. Amino acid sequences of mammalian IGF-I and IGF-II, suchas the mouse and human proteins, are available on the internet throughGENBANK®, see for example GENBANK® Accession No. CAA00082 (human IGF-II,Jan. 28, 1993), AAB21519 (human IGF-II, May 17, 2002), NP_(—)034644(mouse IGF-II, updated Aug. 6, 2006) NP_(—)034642 (mouse IGF-I, updatedAug. 6, 2006), which are incorporated herein by reference. The aminoacid sequence of the insulin receptor is available through GENBANK®, seeAccession Nos. P6213 (Jan. 1, 1998) and NP000199 (Apr. 19, 2006).

Binding of IGFs to IGF-IR activates its intracellular tyrosine kinasedomain, which results in autophosphorylation of the receptor. This inturn results in activation of various pathways that serve to increasecell proliferation, cell motility, and protection from apoptosis. IGF-IRhas been linked to increased growth, survival, and oncogenictransformation of cancer cells (Kaleko et al., Mol Cell Biol 10:464-473,1990; Baserga et al., Biochim Biophys Acta 1332:F105-F126, 1997;Blakesley et al., J Endocrinol 152:339-344, 1997; Khandwala et al.,Endocr Rev 21:215-244, 2000), and overexpression of IGF-IR has beenobserved in a variety of tumor types (Bergmann et al., Cancer Res55:2007-2011, 1995; Werner et al., Adv Cancer Res 68:183-223, 1996;Happerfield et al., J Pathol 183:412-417, 1997; Xie et al., Cancer Res59:3588-3591, 1999; Khandwala et al., Endocr Rev 21:215-244, 2000;Hellawell et al., Cancer Res 62:2942-2950, 2002; Weber et al., Cancer95:2086-2095, 2002). The ligands of IGF-IR, IGF-I and IGF-II, are knownto functions as mitogens in a variety of cancer cell lines (Cullen etal., Cancer Res 50:48-53, 1990; Ankrapp et al., Cancer Res 53:3399-3404,1993; Kappel et al., Cancer Res 54:2803-2807 1994; Guo et al., J Am CollSurg 181:145-154, 1995; Steller et al., Cancer Res 56:1761-1765, 1996;Hermanto et al., Cell Growth Differ 11:655-664, 2000). Many tumorsoverexpress the IGF-II ligand (Werner et al., Adv Cancer Res 68:183-223,1996), exhibiting IGF-II expression levels several fold higher thanthose of IGF-I.

Disclosed herein are human monoclonal antibodies that specifically bindhuman IGF-I and human IGF-II with very high affinity. In someembodiments, the antibody inhibits phosphorylation of the insulin-likegrowth factor receptor. In additional embodiments, the antibody inhibitsthe phosphorylation of the insulin receptor. In additional examples, theantibody inhibits phosphorylation of the insulin-like growth factorreceptor. In a further embodiment, administration of an effective amountof the antibody to a subject decreases the autophosphorylation ontyrosine residues of the human IGF-IR as compared to a control. Thephosphorylation of the human IFG-1R can be measured by any method knownto one of skill in the art.

In one embodiment, the antibodies bind IGF-I and IGF-II with anequilibrium constant (K_(d)) of 200 pM or less. In other embodiments,the antibodies bind IGF-I and IGF-II with an equilibrium associationconstant (K_(d)) of 100 pM or less. In further embodiments, theantibodies bind IGF-I and IGF-II with an equilibrium constant (K_(d)) of60 pM or less. In further embodiments, the antibodies bind IGF-I with anequilibrium constant (K_(d)) of 200 pM or less and IGF-II with anequilibrium constant (K_(d)) of 60 pM or less. In specific examples, thedisclosed antibodies bind IGF-I and IGF-II with a high binding affinity,such as at least about 10 pm, at least about 50 pM, at least about 60pM, at least about 100 pM, at least about 200 pM or about least about500 pM for IGF-I and/or IGF-II. Affinity can be measured by surfaceplasmin resonance.

In some embodiments, the antibodies have a K_(D) for IGF-I and IGF-II of1×10⁻¹⁰ M or less. In other embodiments, the antibodies have a K_(D) forIGF-I and IGF-II of about 1.5×10⁻¹⁰, about 2.0×10⁻¹⁰, about 2.5×10⁻¹⁰,about 3.0×10⁻¹⁰, about 3.5×10⁻¹⁰, about 4.0×10⁻¹⁰, about 4.5×10⁻¹⁰, orabout 5.0×10⁻¹⁰ M or less. In further embodiments, the antibodies have aK_(D) for IGF-I and IGF-II of about 1.5×10⁻¹¹, about 2.0×10⁻¹¹, about2.5×10⁻¹¹, about 3.0×10⁻¹¹, about 3.5×10⁻¹¹, about 4.0×10⁻¹¹, about4.5×10⁻¹¹, or about 5.0×10⁻¹¹ M or less. In additional embodiments, a,the antibodies have a K_(D) for IGF-I and IGF-II of about 1.5×10⁻¹²,about 2.0×10⁻¹², about 2.5×10⁻¹², about 3.0×10⁻¹², about 3.5×10⁻¹²,about 4.0×10⁻¹², about 4.5×10⁻¹², or about 5.0×10⁻¹² M or less. Inseveral embodiments, the human monoclonal antibodies bind human IGF-Iwith a binding affinity of 1×10⁻¹⁰ M⁻¹, at least about 2×10⁻¹⁰M⁻¹, atleast about 3×10⁻¹⁰ M⁻¹, at least about 1×10⁻¹¹ M⁻¹, at least about2.0×10⁻¹¹ M⁻¹, at least about 3×10⁻¹¹ M⁻¹ at least about 6×10⁻¹¹ M⁻¹, orat least about 8×10⁻¹¹ M⁻¹. In additional embodiments, the humanmonoclonal antibodies bind human IGF-II with a binding affinity of1×10⁻¹⁰ M⁻¹, at least about 2×10⁻¹⁰ M⁻¹, at least about 3×10⁻¹⁰ M⁻¹, atleast about 1×10⁻¹¹ M⁻¹, at least about 2.0×10⁻¹¹ M⁻¹, at least about3×10⁻¹¹ M⁻¹ at least about 6×10⁻¹¹ M⁻¹, or at least about 8×10⁻¹¹ M⁻¹.

In additional examples, the human monoclonal antibody binds the epitopeof IGF-II bound by m708.5, which is disclosed herein. Thus, in oneexample, the human monoclonal antibody binds the epitope of IGF-I andIGF-II bound m708.5.

A major limitation in the clinical use of mouse monoclonal antibodies isthe development of a human anti-murine antibody (HAMA) response in thepatients receiving the treatments. The HAMA response can involveallergic reactions and an increased rate of clearance of theadministered antibody from the serum. Various types of modifiedmonoclonal antibodies have been developed to minimize the HAMA responsewhile trying to maintain the antigen binding affinity of the parentmonoclonal antibody. One type of modified monoclonal antibody is ahuman-mouse chimera in which a murine antigen-binding variable region iscoupled to a human constant domain (Morrison and Schlom, ImportantAdvances in Oncology, Rosenberg, S. A. (Ed.), 1989). A second type ofmodified monoclonal antibody is the complementarity determining region(CDR)-grafted, or humanized, monoclonal antibody (Winter and Harris,Immunol. Today 14:243-246, 1993). In some embodiments, the antibodiesdisclosed herein are fully human; both the framework region and the CDRsare from human antibodies. Thus, a HAMA is not induced when these humanantibodies are administered to human subjects.

In several embodiments, the human monoclonal antibody includes at leastone of the light chain CDRs and/or at least one of the heavy chain CDRsfrom the variable heavy and light chain sequences shown below:

Consensus V_(H) (SEQ ID NO: 7)QVQLQQX₁GAEVKMPGSSVKX₂SCX₃ASGGTFSSYAISWX₄RQAPGQGLEWMGGIIPTLX₅IVKYX₆X₇KFQGRVTITADX₈SX₉X₁₀TX₁₁YMELSX₁₂LX₁₃SEDTAVYYCAGGPRGYSYNFDX₁₄ WX₁₅QGTX₁₆VTVSSwherein X₁ is L or P; X₂ is V or I; X₃ is K or R; X₄ is V or M; X₅ is Gor S; X₆ is A or S; X₇ is Q or P; X₈ is K or E; X₆ is T or K; X₁₀ is Sor G; X₁₁ is A or V; X₁₂ is S or N; X₁₃ is G or R; X₁₄ is N or E; X₁₅ isG or S; and X₁₆ is L or M and wherein H-CDR1 is amino acids 26-33 of SEQID NO: 7, H-CDR2 is amino acids 51-58 of SEQ ID NO: 7, and H-CDR3 isamino acids 97-109 of SEQ ID NO: 7. The locations of the CDRs areunderlined in the sequence shown above.

Consensus VL (SEQ ID NO: 8)DIQX₁₇TQSPSSLSASVGDRVTIX₁₈CRASQTISRYX₁₉NWYQQKPGKAPKLLIYAASX₂₀LQSGX₂₁SSRFSGSGSGTEFX₂₂LTISSLQPEDFATYFCQQTYSPPITFGQGTRLEIKX₂₃wherein X₁₇ is M or I; X₁₈ is V or A; X₁₉ is V or L; X₂₀ is S or N; X₂₁is V or I; X₂₂ is A or T; and X₂₃ is Q or R and wherein L-CDR1 is aminoacids 27-32 of SEQ ID NO: 8, L-CDR2 is amino acids 50-52 of SEQ ID NO:8, and L-CDR3 is amino acids 89-97 of SEQ ID NO: 8. The locations of theCDRs are underlined in the sequence shown above.

In one embodiment, the variable region of the heavy chain of the humanmonoclonal antibody includes amino acids 97-109 of SEQ ID NO: 7 (HCDR3).The heavy chain of the isolated human monoclonal antibody can includeone or more of amino acids 26-33 of SEQ ID NO: 7 (HCDR1) and/or aminoacids 51-58 of SEQ ID NO: 7 (HCDR2) and/or amino acids 97-109 of SEQ IDNO: 7 (HCDR3), or all of these sequences. Thus, the heavy chain caninclude amino acids 26-33 of SEQ ID NO: 7 (HCDR1) and amino acids 51-58of SEQ ID NO: 7 (HCDR2) and amino acids 97-109 of SEQ ID NO: 7 (HCDR3).

In some examples, X₁₄ is E. Thus, the variable region of the heavy chaincan include amino acids 97-109 of SEQ ID NO: 7 (HCDR3), wherein X₁₄ isE. The heavy chain of the isolated human monoclonal antibody can includeone or more of amino acids 26-33 of SEQ ID NO: 7 (HCDR1) and/or aminoacids 51-58 of SEQ ID NO: 7 (HCDR2) and/or amino acids 97-109 of SEQ IDNO: 7 (HCDR3) wherein X₁₄ is E, or all of these sequences. Thus, theheavy chain can include amino acids 26-33 of SEQ ID NO: 7 (HCDR1) andamino acids 51-58 of SEQ ID NO: 7 (HCDR2) and amino acids 97-109 of SEQID NO: 7 (HCDR3) wherein X₁₄ is E.

In other examples, X₁₄ is N. The heavy chain of the isolated humanmonoclonal antibody can include one or more of amino acids 26-33 of SEQID NO: 7 (HCDR1) and/or amino acids 51-58 of SEQ ID NO: 7 (HCDR2) and/oramino acids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₁₄ is N, or all ofthese sequences. Thus, the heavy chain can include amino acids 26-33 ofSEQ ID NO: 7 (HCDR1) and amino acids 51-58 of SEQ ID NO: 7 (HCDR2) andamino acids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₁₄ is N.

In further examples, X₅ is G. The heavy chain of the isolated humanmonoclonal antibody can include one or more of amino acids 26-33 of SEQID NO: 7 (HCDR1) and/or amino acids 51-58 of SEQ ID NO: 7 (HCDR2) and/oramino acids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₅ is G, or all ofthese sequences. Thus, the heavy chain can include amino acids 26-33 ofSEQ ID NO: 7 (HCDR1) and amino acids 51-58 of SEQ ID NO: 7 (HCDR2) andamino acids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₅ is G.

In other examples, X₅ is S. The heavy chain of the isolated humanmonoclonal antibody can include one or more of amino acids 26-33 of SEQID NO: 7 (HCDR1) and/or amino acids 51-58 of SEQ ID NO: 7 (HCDR2) and/oramino acids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₅ is S, or all ofthese sequences. Thus, the heavy chain can include amino acids 26-33 ofSEQ ID NO: 7 (HCDR1), amino acids 51-58 of SEQ ID NO: 7 (HCDR2) andamino acids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₅ is S.

In yet additional examples, such as m708.5, X₅ is G and X₁₄ is N. Theheavy chain of the isolated human monoclonal antibody can include one ormore of amino acids 26-33 of SEQ ID NO: 7 (HCDR1) and/or amino acids51-58 of SEQ ID NO: 7 (HCDR2) and/or amino acids 97-109 of SEQ ID NO: 7(HCDR3) wherein X₅ is G and X₁₄ is N, or all of these sequences. Thus,the heavy chain can include amino acids 26-33 of SEQ ID NO: 7 (HCDR1)and amino acids 51-58 of SEQ ID NO: 7 (HCDR2) and amino acids 97-109 ofSEQ ID NO: 7 (HCDR3) wherein X₅ is G and X₁₄ is N. In further examples,such as m708.7, X₅ is G and X₁₄ is E. The heavy chain of the isolatedhuman monoclonal antibody can include one or more of amino acids 26-33of SEQ ID NO: 7 (HCDR1) and/or amino acids 51-58 of SEQ ID NO: 7 (HCDR2)and/or amino acids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₅ is G andX₁₄ is E, or all of these sequences. Thus, the heavy chain can includeamino acids 26-33 of SEQ ID NO: 7 (HCDR1) and amino acids 51-58 of SEQID NO: 7 (HCDR2) and amino acids 97-109 of SEQ ID NO: 7 (HCDR3) whereinX₅ is G and X₁₄ is E. In other examples, such as m708.6, X₅ is S and X₁₄is E. The heavy chain of the isolated human monoclonal antibody caninclude one or more of amino acids 26-33 of SEQ ID NO: 7 (HCDR1) and/oramino acids 51-58 of SEQ ID NO: 7 (HCDR2) and/or amino acids 97-109 ofSEQ ID NO: 7 (HCDR3) wherein X₅ is S and X₁₄ is E, or all of thesesequences. Thus, the heavy chain can include amino acids 26-33 of SEQ IDNO: 7 (HCDR1) and amino acids 51-58 of SEQ ID NO: 7 (HCDR2) and aminoacids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₅ is S and X₁₄ is E.

The light chain of the variable region of the human monoclonal antibodycan include amino acids 27-32 of SEQ ID NO: 8 (LCDR1). The variableregion of the light chain of the human monoclonal antibody can includeamino acids 27-32 of SEQ ID NO: 8 (LCDR1), amino acids 50-52 of SEQ IDNO: 8 (LCDR2) and/or amino acids 89-97 of SEQ ID NO: 8 (LCDR3), or allof these sequences.

In yet additional examples, such as m708.5, X₅ is G and X₁₄ is N. Theheavy chain of the isolated human monoclonal antibody includes aminoacids 26-33 of SEQ ID NO: 7 (HCDR1) and amino acids 51-58 of SEQ ID NO:7 (HCDR2) and amino acids 97-109 of SEQ ID NO: 7 (HCDR3) wherein X₅ is Gand X₁₄ is N. The light chain of the isolated human monoclonal antibodyincludes amino acids 27-32 of SEQ ID NO: 8 (LCDR1), amino acids 50-52 ofSEQ ID NO: 8 (LCDR2) and amino acids 89-97 of SEQ ID NO: 8 (LCDR3).

In further examples, such as m708.7, X₅ is G and X₁₄ is E. The heavychain includes amino acids 26-33 of SEQ ID NO: 7 (HCDR1) and amino acids51-58 of SEQ ID NO: 7 (HCDR2) and amino acids 97-109 of SEQ ID NO: 7(HCDR3) wherein X₅ is G and X₁₄ is E. The light chain of the isolatedhuman monoclonal antibody includes amino acids 27-32 of SEQ ID NO: 8(LCDR1), amino acids 50-52 of SEQ ID NO: 8 (LCDR2) and amino acids 89-97of SEQ ID NO: 8 (LCDR3).

In other examples, such as m708.6, X₅ is S and X₁₄ is E. The heavy chainincludes amino acids 26-33 of SEQ ID NO: 7 (HCDR1) and amino acids 51-58of SEQ ID NO: 7 (HCDR2) and amino acids 97-109 of SEQ ID NO: 7 (HCDR3)wherein X₅ is S and X₁₄ is E. The light chain of the isolated humanmonoclonal antibody includes amino acids 27-32 of SEQ ID NO: 8 (LCDR1),amino acids 50-52 of SEQ ID NO: 8 (LCDR2) and amino acids 89-97 of SEQID NO: 8 (LCDR3).

In several examples, the human monoclonal antibody includes at least oneof the light chain CDRs and/or at least one of the heavy chain CDRs fromthe variable heavy and light chain sequences shown below:

>m708.5 Heavy chain variable domainQVQLQQLGAEVKMPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPTLGIVKYAQKFQGRVTITADKSTSTAYMELSSLGSEDTAVYYCAGGPRGYSYNFDNWGQGTLVTVSS,(SEQ ID NO: 7, wherein X₁is L; X₂ is V; X₃ is K; X₄ is V; X₅ is G; X₆ is A;X₇ is Q; X₈ is K; X₉ is T; X₁₀ is S; X₁₁ is A; X₁₂is S; X₁₃ is G; X₁₄ is N; X₁₅ is G; and X₁₆ is L)wherein H-CDR1 is amino acids 26-33 of SEQ ID NO: 7, H-CDR2 is aminoacids 51-58 of SEQ ID NO: 7, and H-CDR3 is amino acids 97-109 of SEQ IDNO: 7. The locations of the CDRs are highlighted and underlined in thesequence shown above.

Light chain variable domainDIQMTQSPSSLSASVGDRVTIVCRASQTISRYVNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFALTISSLQPEDFATYFCQQTYSPPITFGQGTRLEIKQ, (SEQ ID NO: 8, wherein X₁₇ is M; X₁₈is V; X₁₉ is V; X₂₀ is S; X₂₁ is V; X₂₂ is A; and X₂₃ is Q)wherein L-CDR1 is amino acids 27-32 of SEQ ID NO: 8, L-CDR2 is aminoacids 50-52 of SEQ ID NO: 8, and L-CDR3 is amino acids 89-97 of SEQ IDNO: 8. The locations of the CDRs are highlighted and underlined in thesequence shown above.

In one example, the heavy chain of the monoclonal antibody includes theamino acid sequence set forth as SEQ ID NO: 7, wherein X₁ is L; X₂ is V;X₃ is K; X₄ is V; X₅ is G; X₆ is A; X₇ is Q; X₈ is K; X₉ is T; X₁₀ is S;X₁₁ is A; X₁₂ is S; X₁₃ is G; X₁₄ is N; X₁₅ is G; and X₁₆ is L. Inanother example, the light chain of the monoclonal antibody includes theamino acid sequence set forth as SEQ ID NO: 8, wherein X₁₇ is M; X₁₈ isV; X₁₉ is V; X₂₀ is S; X₂₁ is V; X₂₂ is A; and X₂₃ is Q. In a furtherexample, the heavy chain of the monoclonal antibody includes the aminoacid sequence set forth as SEQ ID NO: 7, wherein X₁ is L; X₂ is V; X₃ isK; X₄ is V; X₅ is G; X₆ is A; X₇ is Q; X₈ is K; X₉ is T; X₁₀ is S; X₁₁is A; X₁₂ is S; X₁₃ is G; X₁₄ is N; X₁₅ is G; and X₁₆ is L and the lightchain of the monoclonal antibody includes the amino acid sequence setforth as SEQ ID NO: 8, wherein X₁₇ is M; X₁₈ is V; X₁₉ is V; X₂₀ is S;X₂₁ is V; X₂₂ is A; and X₂₃ is Q.

However, in other examples, the heavy chain of the monoclonal antibodyincludes the amino acid sequence set forth as SEQ ID NO: 7, wherein X₁is P; X₂ is V; X₃ is K; X₄ is M; X₅ is S; X₆ is A; X₇ is P; X₈ is K; X₉is T; X₁₀ is G; X₁₁ is A; X₁₂ is N; X₁₃ is R; X₁₄ is E; X₁₅ is S; andX₁₆ is M. In another example, the light chain of the monoclonal antibodyincludes the amino acid sequence set forth as SEQ ID NO: 8, wherein X₁₇is I; X₁₈ is A; X₁₉ is L; X₂₀ is S; X₂₁ is V; X₂₂ is T; and X₂₃ is R. Ina further example, the heavy chain of the monoclonal antibody includesthe amino acid sequence set forth as SEQ ID NO: 7, wherein X₁ is P; X₂is V; X₃ is K; X₄ is M; X₅ is S; X₆ is A; X₇ is P; X₈ is K; X₉ is T; X₁₀is G; X₁₁ is A; X₁₂ is N; X₁₃ is R; X₁₄ is E; X₁₅ is S; and X₁₆ is M andthe light chain of the monoclonal antibody includes the amino acidsequence set forth as SEQ ID NO: 8, wherein X₁₇ is I; X₁₈ is A; X₁₉ isL; X₂₀ is 5; X₂₁ is V; X₂₂ is T; and X₂₃ is R.

In yet other examples, the heavy chain of the monoclonal antibodyincludes the amino acid sequence set forth as SEQ ID NO: 7, wherein X₁is P; X₂ is I; X₃ is R; X₄ is V; X₅ is G; X₆ is S; X₇ is Q; X₈ is E; X₉is K; X₁₀ is S; X₁₁ is V; X₁₂ is S; X₁₃ is R; X₁₄ is E; X₁₅ is S; andX₁₆ is L. In another example, the light chain of the monoclonal antibodyincludes the amino acid sequence set forth as SEQ ID NO: 8, wherein X₁₇is M; X₁₈ is A; X₁₉ is L; X₂₀ is N; X₂₁ is I; X₂₂ is T; and X₂₃ is R. Infurther examples, the heavy chain of the monoclonal antibody includesthe amino acid sequence set forth as SEQ ID NO: 7, wherein X₁ is P; X₂is I; X₃ is R; X₄ is V; X₅ is G; X₆ is S; X₇ is Q; X₈ is E; X₉ is K; X₁₀is S; X₁₁ is V; X₁₂ is S; X₁₃ is R; X₁₄ is E; X₁₅ is S; and X₁₆ is L andthe light chain of the monoclonal antibody includes the amino acidsequence set forth as SEQ ID NO: 8, wherein X₁₇ is M; X₁₈ is A; X₁₉ isL; X₂₀ is N; X₂₁ is I; X₂₂ is T; and X₂₃ is R.

The amino acid sequence of the heavy chain variable domain of thepresent disclosed antibodies differs from the heavy chain variabledomain of m708.2. In some embodiments, the amino acid sequence of thelight chain variable domain of the presently disclosed antibodiesdiffers from the light chain variable domain of m7082.

Heavy chain variable domain (SEQ ID NO: 9)QVQLQQPGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPILGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR GPRGYSYNFDYWGQGTLVTVSSwherein H-CDR1 is amino acids 26-33 of SEQ ID NO: 9, H-CDR2 is aminoacids 51-58 of SEQ ID NO: 9, and H-CDR3 is amino acids 97-109 of SEQ IDNO: 9. The locations of the CDRs are highlighted and underlined in thesequence shown above.

Light chain variable domain (SEQ ID NO: 10)DIQMTQSPSSLSASVGDRVTIACRASQTISRYLNWYQQKPGKAPKLLIYAASSLQSGVSSRFSGSGSGTEFTLTISSLQPEDFATYFCQQTYSPPITF GQGTRLEIKRwherein L-CDR1 is amino acids 27-32 of SEQ ID NO: 10, L-CDR2 is aminoacids 50-52 of SEQ ID NO: 10, and L-CDR3 is amino acids 89-97 of SEQ IDNO: 10. The locations of the CDRs are highlighted and underlined in thesequence shown above.

In some embodiments, the amino acid sequence of H-CDR2 of the antibodiesis at least two amino acids different than amino acids 51-58 SEQ ID NO:9. In additional embodiments, the H-CDR3 of the antibody is at least twoamino acids different than amino acids 97-109 of SEQ ID NO: 9. In someembodiments, the amino acid sequence of H-CDR2 of the antibodies differsby two amino acids from amino acids 51-58 SEQ ID NO: 9 and the H-CDR3differs by two amino acids from amino acids 97-109 of SEQ ID NO: 9. Infurther embodiments, the antibody does not include the amino acidsequence set forth as SEQ ID NO: 9. In additional embodiments, theantibody does not include amino acids 51-58 of SEQ ID NO: 9 and/or aminoacids 97-109 of SEQ ID NO: 9. In additional embodiments, the antibodydoes not include the amino acid sequence set forth as SEQ ID NO: 9. Inyet other embodiments, the antibody does not include the amino acidsequence set forth as SEQ ID NO: 10.

The monoclonal antibody can be of any isotype. The monoclonal antibodycan be, for example, an IgM or an IgG antibody, such as IgG₁, IgG₂, IgG₃or IgG₄. The class of an antibody that specifically binds IGF-II can beswitched with another. In one aspect, a nucleic acid molecule encodingV_(L) or V_(H) is isolated using methods well-known in the art, suchthat it does not include any nucleic acid sequences encoding theconstant region of the light or heavy chain, respectively. The nucleicacid molecule encoding V_(L) or V_(H) is then operatively linked to anucleic acid sequence encoding a C_(L) or C_(H) from a different classof immunoglobulin molecule. This can be achieved using a vector ornucleic acid molecule that comprises a C_(L) or C_(H) chain, as known inthe art. For example, an antibody that specifically binds IGF-I andIGF-II that was originally IgM may be class switched to an IgG. Classswitching can be used to convert one IgG subclass to another, such asfrom IgG₁ to IgG₂ or to IgG₄.

Fully human monoclonal antibodies include a human framework region. Thishuman framework region can be the framework regions disclosed in one ormore of SEQ ID NOS: 7-8 (these sequences include CDR sequences as wellas framework sequences). Exemplary heavy chain framework regions areamino acids 1-25, 34-50, 59-96, and 110-120 or SEQ ID NO: 7. Thus,exemplary heavy chain framework regions are amino acids 1-25, 34-50,59-96, and 110-120 of SEQ ID NO: 7, wherein X₁ is L; X₂ is V; X₃ is K;X₄ is V; X₆ is A; X₇ is Q; X₈ is K; X₉ is T; X₁₀ is S; X₁₁ is A; X₁₂ isS; X₁₃ is G; X₁₅ is G; and X₁₆ is L. Exemplary heavy chain frameworkregions are amino acids 1-25, 34-50, 59-96, and 110-120 of SEQ ID NO: 7,wherein X₁ is P; X₂ is V; X₃ is K; X₄ is M; X₆ is A; X₇ is P; X₈ is K;X₉ is T; X₁₀ is G; X₁₁ is A; X₁₂ is N; X₁₃ is R; X₁₅ is S; and X₁₆ is M.Additional exemplary heavy chain framework regions are amino acids 1-25,34-50, 59-96, and 110-120 of SEQ ID NO: 7, wherein X₁ is P; X₂ is I; X₃is R; X₄ is V; X₆ is S; X₇ is Q; X₈ is E; X₉ is K; X₁₀ is S; X₁₁ is V;X₁₂ is S; X₁₃ is R; X₁₅ is S; and X₁₆ is L. Exemplary light chainframework regions are amino acids 1-26, 33-49, 53-88 and 98-108 of SEQID NO: 8. wherein X₁₇ is M; X₁₈ is V; X₁₉ is V; X₂₀ is S; X₂₁ is V; X₂₂is A; and X₂₃ is Q. Exemplary light chain framework regions are aminoacids 1-26, 33-49, 53-88 and 98-108 of SEQ ID NO: 8. wherein X₁₇ is I;X₁₈ is A; X₁₉ is L; X₂₀ is S; X₂₁ is V; X₂₂ is T; and X₂₃ is R.Additional exemplary light chain framework regions are amino acids 1-26,33-49, 53-88 and 98-108 of SEQ ID NO: 8. wherein X₁₇ is M; X₁₈ is A; X₁₉is L; X₂₀ is N; X₂₁ is I; X₂₂ is T; and X₂₃ is R. Exemplary frameworkregions are shown in FIG. 10, and include the framework regions fromm708.5, m708.6 and m708.7.

However, the framework regions can be from another source. Exemplaryframework regions are disclosed, for example, in U.S. Pat. No.7,824,681, which is incorporated herein by reference.

In some embodiments, the light chain of the antibody includes aminoacids 1-25, amino acids 34-50, amino acids 59-96 and/or amino acids110-120 of SEQ ID NO: 7. In additional embodiments, the light chain ofthe antibody includes amino acids 1-25, amino acids 34-50, amino acids59-96 and/or amino acids 110-120 of SEQ ID NO: 9. In other embodiments,the antibody includes amino acids 1-26, amino acids 33-49, amino acids53-88 and/or amino acids 98-108 of SEQ ID NO: 8. In further embodiments,the antibody includes amino acids 1-26, amino acids 33-49, amino acids53-88 and/or amino acids 98-108 of SEQ ID NO: 10.

The antibodies disclosed herein can include a light chain frameworkregion and a heavy chain framework region. Thus, the antibody caninclude all of the light chain framework regions (amino acids of theamino acid sequence set forth as SEQ ID NO: 8 or the amino acid sequenceset forth as SEQ ID NO: 10. The antibody can include all of the heavychain framework regions of the amino acid sequence set forth as SEQ IDNO: 7 or the amino acid sequence set forth as SEQ ID NO: 9.

In some embodiments, the antibody includes a light chain frameworkregion, but do not include the light chain framework regions of theamino acid sequence set forth as SEQ ID NO: 8. In additionalembodiments, the antibody includes a heavy chain framework region, butdo not include the heavy chain framework region regions of the aminoacid sequence set forth as SEQ ID NO: 10. In further embodiments, theantibody includes the heavy chain framework regions of the amino acidsequence set forth as SEQ ID NO: 7 with at most 1, 2, 3 or 4conservative substitutions and the light chain framework region of theamino acid sequence set forth as SEQ ID NO: 8 with at most 1, 2, 3, or 4conservative substitutions. The antibody

Antibody fragments that specifically bind IGF-I and IGF-II areencompassed by the present disclosure, such as Fab, F(ab′)₂, and Fvwhich include a heavy chain and light chain variable region and arecapable of binding the epitopic determinant on IGF-I and IGF-II. Theseantibody fragments retain the ability to selectively bind with theantigen. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of anscFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). In several examples, the variable regionincluded in the antibody is the variable region of m708.5. In one groupof embodiments, the antibodies have V_(H) CDRs of m708.5, or acombination of these CDRs, as discussed above. In one group ofembodiments, the antibodies have V_(L) CDRs of m708.5, or a combinationof these CDRs, as discussed above.

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds. In several embodiments, the antibodies disclosed herein areactive in an scFV format.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of asingle chain antibody (scFV₂), are also contemplated. In severalembodiments, the antibodies disclosed herein are active in an scFVformat.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced, that retain the ability to bind to IGF-I and IGF-IIwith picomolar affinity. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules. Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Thus, one of skill in the art can readily review the sequences of theantibodies disclosed herein, such and the framework regions, andidentify a conservative substitution, and produce the conservativevariant using well-known molecular techniques.

Effector molecules, such as therapeutic, diagnostic, or detectionmoieties can be linked to an antibody of interest, such as a humanantibody that specifically binds IGF-I and IGF-II with picomolaraffinity, using any number of means known to those of skill in the art.Both covalent and noncovalent attachment means may be used. Theprocedure for attaching an effector molecule to an antibody variesaccording to the chemical structure of the effector. Polypeptidestypically contain a variety of functional groups; such as carboxylicacid (COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on an antibodyto result in the binding of the effector molecule. Alternatively, theantibody is derivatized to expose or attach additional reactivefunctional groups. The derivatization may involve attachment of any of anumber of linker molecules such as those available from Pierce ChemicalCompany, Rockford, Ill. The linker can be any molecule used to join theantibody to the effector molecule. The linker is capable of formingcovalent bonds to both the antibody and to the effector molecule.Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. Where theantibody and the effector molecule are polypeptides, the linkers may bejoined to the constituent amino acids through their side groups (such asthrough a disulfide linkage to cysteine) or to the alpha carbon aminoand carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (such as enzymes or fluorescent molecules) drugs,toxins, and other agents to antibodies one skilled in the art will beable to determine a suitable method for attaching a given agent to anantibody or other polypeptide.

The antibodies or any of they antibody fragments disclosed herein thatspecifically bind IGF-I and IGF-II with picomolar affinity can bederivatized or linked to another molecule (such as another peptide orprotein). In general, the antibodies or portion thereof is derivatizedsuch that the binding to IGF-I and IGF-II is not affected adversely bythe derivatization or labeling. For example, the antibody can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (for example, a bispecific antibody or a diabody), adetection agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate associate of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, such as to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (such as disuccinimidyl suberate). Suchlinkers are available from Pierce Chemical Company, Rockford, Ill.Bispecific antibodies can also be produced using linkers of G₄Striplicates.

Thus, bispecific antibodies are provided, wherein the bispecificantibody includes any of the antibodies that specifically bind IGF-I andIGF-II with picomolar affinity. In several embodiments, the bispecificantibody includes the CDRs of the heavy chain variable region of anantibody that specifically binds IGF-II only, and the CDRs of the lightchain variable region of an antibody that specifically binds IGF-IIonly. Antibodies that bind IGF-II are disclosed, for example, in U.S.Provisional Application No. 61/548,164, filed Oct. 17, 2011, which isincorporated herein by reference. Additional antibodies thatspecifically bind IGF-II, such as, but not limited to, m606, m610, m616are disclosed, for example, in PCT Publication No. WO 2007/022172, whichis also incorporated herein by reference. These antibodies can inhibitthe phosphorylation of the IGF-IR and IR, and inhibit the growth andmigration of human cancer cells in vitro and in vivo.

In some embodiments, the bispecific antibody includes a heavy chainvariable region that includes the amino acid sequence set forth as aminoacids 26-33 of SEQ ID NO: 7, amino acids 51-58 of SEQ ID NO: 7 and aminoacids 97-109 of SEQ ID NO: 7 and a light chain variable region thatincludes the amino acid sequence set forth as amino acids 27-32 of SEQID NO: 8, amino acids 50-52 of SEQ ID NO: 8 and amino acids 89-97 of SEQID NO: 8.

In some embodiments, the bispecific antibodies includes the heavy chainvariable region and/or the CDRs of the m610.27 heavy chain variableregion amino acid sequence:

(SEQ ID NO: 23) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSRLRSDDTAVYYCAR DVQWLAYGMDVWGQGTTVTVSS                       Variable regionThus, in some embodiments, the heavy chain variable region of theantibody that specifically binds IGF-II includes one or more of aminoacids 26-33 of SEQ ID NO: 23, amino acids 51-58 of SEQ ID NO: 23, andamino acids 97-109 of SEQ ID NO: 23, and specifically binds IGF-II. Inadditional embodiments, the heavy chain variable region of the antibodythat specifically binds IGF-II includes amino acids 26-33 of SEQ ID NO:23, amino acids 51-58 of SEQ ID NO: 23, and amino acids 97-109 of SEQ IDNO: 23, and specifically binds IGF-II. In further embodiments, the heavychain variable region includes the amino acid sequence set forth as SEQID NO: 23.

In additional embodiments, the bispecific antibody includes the lightchain variable region and/or the CDRs of m610.27 light chain variableregion amino acid sequence:

(SEQ ID NO: 24) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGRAPDLLINAASSLQSGVFSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSYSLPFTF GGGTKVEIK                              Variable regionThus, in some embodiments, the light chain variable region of theantibody that specifically binds IGF-II includes at least one of aminoacids 27-32 of SEQ ID NO: 24, amino acids 50-52 of SEQ ID NO: 24, andamino acids 89-98 of SEQ ID NO: 24, and specifically binds IGF-II. Inadditional embodiments, the light chain variable region of the antibodythat specifically binds IGF-II includes amino acids 27-32 of SEQ ID NO:24, amino acids 50-52 of SEQ ID NO: 24, and amino acids 89-98 of SEQ IDNO: 24, and specifically binds IGF-II. In further embodiments, the lightchain variable region of the bispecific antibody includes the amino acidsequence set forth as SEQ ID NO: 24.

In some embodiments, the bispecific antibody that specifically bindsIGF-II includes a heavy chain variable region comprising amino acids26-33 of SEQ ID NO: 23, amino acids 51-58 of SEQ ID NO: 23, and aminoacids 97-109 of SEQ ID NO: 23, and the light chain variable regioncomprising amino acids 27-32 of SEQ ID NO: 24, amino acids 50-52 of SEQID NO: 24, and amino acids 89-98 of SEQ ID NO: 24. In additionalembodiments, the bispecific antibody that specifically binds IGF-IIincludes a heavy chain variable region comprising the amino acidsequence set forth as SEQ ID NO: 23, and a light chain variable regioncomprising the amino acid sequence set forth as SEQ ID NO: 24.

Thus, in specific non-liming examples, the bispecific antibody includesa heavy chain variable region that includes the amino acid sequence setforth as amino acids 26-33 of SEQ ID NO: 7, amino acids 51-58 of SEQ IDNO: 7 and amino acids 97-109 of SEQ ID NO: 7 a light chain variableregion that includes the amino acid sequence set forth as amino acids27-32 of SEQ ID NO: 8, amino acids 50-52 of SEQ ID NO: 8 and amino acids89-97 of SEQ ID NO: 8. The antibody also includes a heavy chain variableregion comprising amino acids 26-33 of SEQ ID NO: 23, amino acids 51-58of SEQ ID NO: 23, and amino acids 97-109 of SEQ ID NO: 23, and a lightchain variable region comprising amino acids 27-32 of SEQ ID NO: 24,amino acids 50-52 of SEQ ID NO: 24, and amino acids 89-98 of SEQ ID NO:24.

In additional embodiments, the bispecific antibody includes an antigenbinding fragment of the antibody that specifically binds IGF-II onlyand/or an antigen binding fragment of the monoclonal antibody thatspecifically bind both IGF-I and IGF-II with picomolar affinity. Theantigen binding fragment can be an scFv, Fab, F(ab′)₂, or an Fv.

A human antibody (or bispecific form thereof) that specifically bindsIGF-I and IGF-II with high affinity (for example, picomolar affinity),can be labeled with a detectable moiety. Useful detection agents includefluorescent compounds, including fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonylchloride, phycoerythrin, lanthanide phosphors and the like.Bioluminescent markers are also of use, such as luciferase, Greenfluorescent protein (GFP), Yellow fluorescent protein (YFP). An antibodycan also be labeled with enzymes that are useful for detection, such ashorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, glucose oxidase and the like. When an antibody is labeledwith a detectable enzyme, it can be detected by adding additionalreagents that the enzyme uses to produce a reaction product that can bediscerned. For example, when the agent horseradish peroxidase ispresent, the addition of hydrogen peroxide and diaminobenzidine leads toa colored reaction product, which is visually detectable. An antibodymay also be labeled with biotin, and detected through indirectmeasurement of avidin or streptavidin binding. It should be noted thatthe avidin itself can be labeled with an enzyme or a fluorescent label.

An antibody may be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes. Forinstance, the radiolabel may be used to detect IGF-I and/or IGF-II byx-ray, emission spectra, or other diagnostic techniques. Examples oflabels for polypeptides include, but are not limited to, the followingradioisotopes or radionuclides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

In one embodiment, the antibody that specifically binds IGF-I and IGF-IIinhibits phosphorylation of the insulin-like growth factor type Ireceptor (IGF-IR). IGF-II binds the IGF-I receptor, and causes tyrosinephosphorylation. Tyrosine phosphorylation of IGF-IR is one of the earlyresponses to potent mitogenic stimuli, such as the binding of IGF-I orIFG-II. The IGF-I receptor binds IGF-I and IGF-II with high affinity toactivate cellular proliferation in both normal growth and developmentand malignant transformation and has tyrosine kinase activity. IGF-IR ishighly over expressed in most malignant tissues where it functions as ananti-apoptotic agent by enhancing cell survival. Tyrosinephosphorylation status of proteins can be determined usinganti-phosphotyrosine antibodies. In addition, because of the bindingspecificity of the SH2 domain to phosphorylated tyrosine residues, aspecific pattern of tyrosine phosphorylation can be elucidated todetermine phosphorylation status.

Immunoassays for determining IGF-IR tyrosine phosphorylation or formeasuring total IGF-IR levels are an ELISA or Western blot. If only thecell surface level of IGF-IR is to be measured, the cells are not lysed,and the cell surface levels of IGF-IR are measured using one of theassays described herein. In one example, the immunoassay for determiningcell surface levels of IGF-IR includes the steps of labeling the cellsurface proteins with a detectable label, such as ³²P,immunoprecipitating the IGF-IR with an anti-IGF-IR antibody and thendetecting the phosphorylated IGF-IR.

Nucleic acids encoding the amino acid sequences of the antibodies thatbind IGF-II are also provided herein. The nucleic acid molecules canencode a heavy chain variable domain and/or a light chain variabledomain. Exemplary nucleic acid sequences are as follows:

Heavy chain variable domain (m708.5) (SEQ ID NO: 11)CAGGTACAGCTGCAGCAACTAGGGGCTGAAGTGAAGATGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGTAGTTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTACCCTTGGTATAGTAAAGTACGCGCAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGGGATCTGAGGACACGGCCGTGTATTACTGTGCGGGAGGCCCTAGGGGATACAGCTATAACTTTGACAACTGGGGTCAGGGCACCCT GGTCACCGTCTCCTCALight chain variable domain (m708.5) (SEQ ID NO: 12)GACATCCAAATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCGTTTGCCGGGCAAGTCAGACCATTAGTAGGTATGTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCTCATCAAGGTTCAGTGGTAGTGGATCAGGGACAGAGTTCGCTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTATTTCTGTCAACAGACTTACAGTCCCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACAAExemplary nucleic acid sequences are also as follows:

m708.6 Heavy chain variable domain (SEQ ID NO: 19)CAGGTACAGTTGCAACAACCAGGGGCTGAAGTGAAGATGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGATGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGGATCATCCCTACCCTTAGTATAGTAAAGTACGCACCGAAGTTCCAGGGCAGAGTCACGATTACCGCAGACAAATCCACGGGCACAGCCTACATGGAGCTGAGCAACCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGGGAGGCCCTAGGGGATACAGCTATAACTTTGACGAATGGAGTCAGGGCACCAT GGTCACCGTCTCCTCALight chain variable domain (SEQ ID NO: 20)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCGCTTGCCGGGCAAGTCAGACCATTAGTAGGTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAACCAGTTTGCAAAGTGGGGTCTCATCAAGGTTCAGTGGCAGTGGATCTGAGACAGAGTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTATTTCTGTCAACAGACTTACAGTCCCCCGATCACCTTCGGCCAAGGGACACGATTGGAGATTAAACGA m708.7 Heavy chain variable domain(SEQ ID NO: 21) CAGGTACAGTTGCAGCAACCAGGGGCTGAAGTGAAGATGCCTGGGTCCTCGGTGAAGATCTCCTGTAGGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGTCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTACCCTTGGTATAGTAAAGTACTCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCAAGAGCACAGTCTACATGGAACTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGGGAGGCCCTAGGGGATACAGCTATAACTTTGACGAATGGAGTCAGGGCACCCT GGTCACCGTCTCCTCALight chain variable domain (SEQ ID NO: 22)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCGCTTGCCGGGCAAGTCAGACCATTAGTAGGTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGATATCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTATTTCTGTCAACAGACTTATAGTCCCCCGATCACCTTCGGTCAAGGGACACGACTGGAGATTAAACGA.In some embodiment the nucleic acid molecules do not include one or bothof SEQ ID NO: 13 and SEQ ID NO: 14, as set forth below:

Heavy chain variable domain (m708.2) (SEQ ID NO: 13)CAGGTACAGCTGCAGCAGCCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCCTTGGTATAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGCCCTAGGGGATACAGCTATAACTTTGACTACTGGGGCCAGGGCACCCT GGTCACCGTCTCCTCALight chain variable domain (m708.2) (SEQ ID NO: 14)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCGCTTGCCGGGCAAGTCAGACCATTAGTAGGTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCTCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTATTTCTGTCAACAGACTTACAGTCCCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAIn some embodiments, nucleic acid molecules are provided that encode abispecific antibody, such as a bispecific antibody including a lightchain and a heavy chain that specifically bind IGF-I and IGF-II, and alight chain and a heavy chain that specifically bind IGF-II only.Exemplary nucleic acid sequences are as follows:

Heavy chain nucleotide sequence (m610.27) (SEQ ID NO: 25):CAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATGTGCAGTGGCTGGCATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTGAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Light chain nucleotide sequence (m610.27)(SEQ ID NO: 26): GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGTAGCTATTTAAATTGGTATCAGCAGAAGCCAGGGAGAGCCCCTGACCTCCTGATCAATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACCGACTTCACTCTCACCATCAGCAGTCTCCAACCTGAAGATTTTGCAACTTACTTCTGTCAACAGAGTTACAGTCTTCCGTTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAGGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGNucleotide molecules encoding the antibodies can readily be produced byone of skill in the art, using the amino acid sequences provided herein,and the genetic code. In addition, one of skill can readily construct avariety of clones containing functionally equivalent nucleic acids, suchas nucleic acids which differ in sequence but which encode the sameeffector molecule (“EM”) or antibody sequence. Thus, nucleic acidsencoding antibodies, conjugates and fusion proteins are provided herein.

Nucleic acid sequences encoding the human antibodies that specificallybind IGF-I and IGF-II, antigen binding fragments thereof, and bispecificforms thereof, can be prepared by any suitable method including, forexample, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids encoding sequences encoding a human antibodythat specifically binds IGF-I and IGF-II with high affinity (such aspicomolar affinity), antigen binding fragments thereof, and bispecificforms thereof, can be prepared by cloning techniques. Examples ofappropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Sambrook et al., supra, Berger and Kimmel (eds.), supra, andAusubel, supra. Product information from manufacturers of biologicalreagents and experimental equipment also provide useful information.Such manufacturers include the SIGMA Chemical Company (Saint Louis,Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway,N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem GenesCorp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),Invitrogen (San Diego, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

In one example, an antibody of use is prepared by inserting the cDNAwhich encodes a variable region from an antibody into a vector whichcomprises the cDNA encoding an effector molecule (EM), such as an enzymeor label. The insertion is made so that the variable region and the EMare read in frame so that one continuous polypeptide is produced. Thus,the encoded polypeptide contains a functional Fv region and a functionalEM region. In one embodiment, cDNA encoding an enzyme is ligated to ascFv so that the enzyme is located at the carboxyl terminus of the scFv.In several examples, cDNA encoding a horseradish peroxidase or alkalinephosphatase, or a polypeptide marker of interest is ligated to a scFv sothat the enzyme (or polypeptide marker) is located at the amino terminusof the scFv. In another example, the label is located at the aminoterminus of the scFv. In a further example, cDNA encoding the protein orpolypeptide marker is ligated to a heavy chain variable region of anantibody, so that the enzyme or polypeptide marker is located at thecarboxyl terminus of the heavy chain variable region. The heavychain-variable region can subsequently be ligated to a light chainvariable region of the antibody using disulfide bonds. In a yet anotherexample, cDNA encoding an enzyme or a polypeptide marker is ligated to alight chain variable region of an antibody, so that the enzyme orpolypeptide marker is located at the carboxyl terminus of the lightchain variable region. The light chain-variable region can subsequentlybe ligated to a heavy chain variable region of the antibody usingdisulfide bonds.

Once the nucleic acids encoding the antibody, labeled antibody,bispecific form, or fragment thereof are isolated and cloned, theprotein can be expressed in a recombinantly engineered cell such asbacteria, plant, yeast, insect and mammalian cells using a suitableexpression vector. One or more DNA sequences encoding the antibody orfragment thereof can be expressed in vitro by DNA transfer into asuitable host cell. The cell may be prokaryotic or eukaryotic. The termalso includes any progeny of the subject host cell. It is understoodthat all progeny may not be identical to the parental cell since theremay be mutations that occur during replication. Methods of stabletransfer, meaning that the foreign DNA is continuously maintained in thehost, are known in the art. Hybridomas expressing the antibodies ofinterest are also encompassed by this disclosure.

Polynucleotide sequences encoding the antibody, labeled antibody,bispecific form, or antigen binding fragment thereof, can be operativelylinked to expression control sequences. An expression control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with theexpression control sequences. The expression control sequences include,but are not limited to appropriate promoters, enhancers, transcriptionterminators, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, maintenance of the correct readingframe of that gene to permit proper translation of mRNA, and stopcodons.

The polynucleotide sequences encoding the antibody, labeled antibody,bispecific form, or antigen binding fragment thereof can be insertedinto an expression vector including, but not limited to a plasmid, virusor other vehicle that can be manipulated to allow insertion orincorporation of sequences and can be expressed in either prokaryotes oreukaryotes. Hosts can include microbial, yeast, insect and mammalianorganisms. Methods of expressing DNA sequences having eukaryotic orviral sequences in prokaryotes are well known in the art. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide can becarried out by conventional means including preparative chromatographyand immunological separations. Once expressed, the antibody, labeledantibody or functional fragment thereof can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.,1982). Substantially pure compositions of at least about 90 to 95%homogeneity are disclosed herein, and 98 to 99% or more homogeneity canbe used for pharmaceutical purposes. Once purified, partially or tohomogeneity as desired, if to be used therapeutically, the polypeptidesshould be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodies,bispecific forms, and functional fragments thereof that are disclosedherein can also be constructed in whole or in part using standardpeptide synthesis. Solid phase synthesis of the polypeptides of lessthan about 50 amino acids in length can be accomplished by attaching theC-terminal amino acid of the sequence to an insoluble support followedby sequential addition of the remaining amino acids in the sequence.Techniques for solid phase synthesis are described by Barany &Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: SpecialMethods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J.Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid PhasePeptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984.Proteins of greater length may be synthesized by condensation of theamino and carboxyl termini of shorter fragments. Methods of formingpeptide bonds by activation of a carboxyl terminal end (such as by theuse of the coupling reagent N,N′-dicylohexylcarbodimide) are well knownin the art.

Recombinant human antibodies that specifically bind IGF-I and IGF-IIwith high affinity, in addition to the anti-IGF-I and IGF-II antibodiesdisclosed herein can be isolated by screening of a recombinantcombinatorial antibody library, preferably a scFv phage display library,prepared using cDNAs of the variable regions of heavy and light chainsprepared from mRNA derived from human lymphocytes. Methodologies forpreparing and screening such libraries are known in the art. There arecommercially available kits for generating phage display libraries (forexample, the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612). There are also other methods and reagents that can be used ingenerating and screening antibody display libraries (see, for example,U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCTPublication No. WO 91/17271; PCT Publication No. WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; Fuchs etal., Bio/Technology 9:1370-1372, 1991; Hay et al., Hum. Antibod.Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989;McCafferty et al., Nature 348:552-554, 1990; Griffiths et al. EMBO J.12:725-734, 1993)

In one embodiment, to isolate additional human antibodies thatspecifically bind IGF-I and IGF-II, such as a human antibody thatspecifically binds IGF-I and IGF-II with high affinity (such as at leastpicomolar affinity), as described herein, is first used to select humanheavy and light chain sequences having similar binding activity towardIGF-I and IGF-II, such as using the epitope imprinting methods disclosedin PCT Publication No. WO 93/06213. The antibody libraries used in thismethod are scFv libraries prepared and screened, using methods such asthose as described in PCT Publication No. WO 92/01047, McCafferty etal., Nature 348:552-554, 1990; and/or Griffiths et al., EMBO J12:725-734, 1993 using human IGF-II as the antigen.

Once initial human variable light chain (V_(L)) and variable heavy chain(V_(H)) segments are selected, “mix and match” experiments, in whichdifferent pairs of the initially selected V_(L) and V_(H) segments arescreened for binding to IGF-I and IGF-II. These assays are performed toselect V_(L)/V_(H) pair combinations of interest. Additionally, toincrease binding affinity of the antibody, the V_(L) and V_(H) segmentscan be randomly mutated, such as within H-CDR3 region or the L-CDR3region, in a process analogous to the in vivo somatic mutation processresponsible for affinity maturation of antibodies during a naturalimmune response. This in vitro affinity maturation can be accomplishedby amplifying V_(H) and V_(L) regions using PCR primers complimentary tothe H-CDR3 or L-CDR3, respectively. In this process, the primers havebeen “spiked” with a random mixture of the four nucleotide bases atcertain positions such that the resultant PCR products encode V_(H) andV_(L) segments into which random mutations have been introduced into theV_(H) and/or V_(L) CDR3 regions. These randomly mutated V_(H) and V_(L)segments can be tested to determine the binding affinity for IGF-I andIGF-II.

Following screening and isolation of an antibody that binds IGF-I andIGF-II with high affinity, such as at least picomolar affinity, from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (forexample, from the phage genome) and subcloned into other expressionvectors by standard recombinant DNA techniques, as described above. Ifdesired, the nucleic acid can be further manipulated to create otherantibody fragments, also as described below. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described above.

Compositions and Therapeutic Methods

Compositions are provided that include one or more of the antibodiesthat specifically bind IGF-I and IGF-II with high affinity, such as atleast picomolar affinity, antigen binding fragments and bispecific formsthat are disclosed herein, and nucleic acids encoding these antibodies,antigen binding fragments and bispecific forms, and a carrier. Thecompositions can be prepared in unit dosage forms for administration toa subject. The amount and timing of administration are at the discretionof the treating physician to achieve the desired purposes. The antibodycan be formulated for systemic or local (such as intra-tumor)administration. In one example, the antibody that specifically bindsIGF-I and IGF-II with at least picomolar affinity, antigen bindingfragment or bispecific form is formulated for parenteral administration,such as intravenous administration.

The compositions for administration can include a solution of theantibody that specifically binds IGF-I and IGF-II with at leastpicomolar affinity, antigen binding fragment or bispecific form, ornucleic acids encoding these molecules, dissolved in a pharmaceuticallyacceptable carrier, such as an aqueous carrier. A variety of aqueouscarriers can be used, for example, buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of antibody in these formulations can vary widely, andwill be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the subject's needs.

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg of antibody per subject per day. Dosagesfrom 0.1 up to about 100 mg per subject per day may be used,particularly if the agent is administered to a secluded site and notinto the circulatory or lymph system, such as into a body cavity or intoa lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa.(1995).

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.5 to 15 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of RITUXAN® in 1997. Antibodies can be administeredby slow infusion, rather than in an intravenous push or bolus. In oneexample, a higher loading dose is administered, with subsequent,maintenance doses being administered at a lower level. For example, aninitial loading dose of 4 mg/kg may be infused over a period of some 90minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kginfused over a 30 minute period if the previous dose was well tolerated.

In another embodiment, the invention provides a method for inhibitingIGF-IR activity by administering an antibody that binds IGF-I and IGF-IIwith high affinity, such as at least picomolar affinity, antigen bindingfragment or bispecific form thereof, or a nucleic acid encoding one ormore of these molecules, to a subject in need thereof. Thus, theantibodies, antigen binding fragments, bispecific forms, and nucleicacids disclosed herein can be used therapeutically. In one example, thesubject is human. The antibody may be administered to a non-human mammalexpressing an IGF-I and/or IGF-II with which the antibody cross-reacts(such as a primate, or a cynomolgus or rhesus monkey). It should benoted that animal models, such as primate models, can be useful forevaluating the therapeutic efficacy of antibodies disclosed herein.

The antibody, antigen binding fragment, bispecific form or nucleic acidmolecule can be administered to a subject having a disease or disordersin which the presence of high levels of IGF-I receptor activity has beenshown to be or is suspected of being either responsible for thepathophysiology of the disease or disorder or is a factor thatcontributes to a worsening of the disease or disorder. Accordingly,inhibition of IGF-I receptor (IGF-IR) activity is expected to alleviatethe symptoms and/or progression of the disorder. Such disorders may beevidenced, for example, by an increase in the levels of IGF-IR on thecell surface or by increased tyrosine autophosphorylation of IGF-IR inthe affected cells or tissues of a subject suffering from the disorder.

The antibodies disclosed herein that specifically binds IGF-I and IGF-IIwith high affinity, such as at least picomolar affinity, antigen bindingfragments. bispecific forms, or nucleic acids can be administered toslow or inhibit the growth of cells, such as tumor cells. In theseapplications, a therapeutically effective amount is administered to asubject in an amount sufficient to inhibit growth of a tumor, or toinhibit a sign or a symptom of the tumor. Suitable subjects may includethose with a tumor that expresses the IGF-I receptor, such as thosesuffering from a sarcoma, leukemia, prostate cancer, lung cancer, breastcancer, colon cancer, stomach cancer, uterine cancer, cervical cancer,esophageal cancer, liver cancer, pancreatic cancer, kidney cancer,thyroid cancer, brain cancer, or an ovarian cancer. In one embodiment, amethod is provided for the treatment of cancer such as brain, squamouscell, bladder, gastric, pancreatic, breast, head, neck, esophageal,prostate, colorectal, lung, renal, kidney, ovarian, gynecological orthyroid cancer.

A method is also provided herein for the treatment of subjects havingmultiple myeloma, liquid tumor, liver cancer, thymus disorder, T-cellmediated auto-immune disease, endocronological disorder, ischemia,neurodegenerative disorder, lung cancer, bone cancer, pancreatic cancer,skin cancer, cancer of the head and neck, cutaneous or intraocularmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, colon cancer, breast cancer, gynecologictumors (such as uterine sarcomas, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina or carcinoma of the vulva), Hodgkin's disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrine system(such as cancer of the thyroid, parathyroid or adrenal glands), sarcomasof soft tissues, cancer of the urethra, cancer of the penis, prostatecancer, chronic or acute leukemia, solid tumors of childhood,lymphocytic lymphomas, cancer of the bladder, cancer of the kidney orureter (such as renal cell carcinoma, carcinoma of the renal pelvis), orneoplasms of the central nervous system (such as primary CNS lymphoma,spinal axis tumors, brain stem gliomas or pituitary adenomas). Inseveral examples, the human antibody that binds IGF-I and IGF-II withpicomolar affinity, antigen binding fragment, or bispecific form, (or anucleic acid encoding one or more of these molecules) is administered toa patient with prostate cancer, glioma or fibrosarcoma. In additionalexamples, a human antibody that binds IGF-I and IGF-II with highaffinity, such as at least picomolar affinity, antigen binding fragment,or bispecific form (or a nucleic acid encoding one or more of thesemolecules) is administered to a subject with lung, breast, prostate orcolon cancer. In other examples, the method causes the tumor not toincrease in weight or volume or to decrease in weight or volume.

Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health. A therapeuticallyeffective amount of the antibody is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Inone example, the amount of the antibody, antigen binding fragment,bispecific form, or nucleic acid is sufficient to inhibitphosphorylation of the IGF-I receptor. These compositions can beadministered in conjunction with another chemotherapeutic agent, eithersimultaneously or sequentially.

Many chemotherapeutic agents are presently known in the art. In oneembodiment, the chemotherapeutic agents is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, anti-survival agents,biological response modifiers, anti-hormones, e.g. anti-androgens, andanti-angiogenesis agents.

Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2)inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II(cyclooxygenase II) inhibitors, can be used in conjunction with acompound of the invention. Examples of useful COX-II inhibitors includeCELEBREX™ (alecoxib), valdecoxib, and rofecoxib. Examples of usefulmatrix metalloproteinase inhibitors are described in PCT Publication No.WO 96/33172 (published Oct. 24, 1996), PCT Publication No. WO 96/27583(published Mar. 7, 1996), European Patent Application No. 97304971.1(filed Jul. 8, 1997), European Patent Application No. 99308617.2 (filedOct. 29, 1999), PCT Publication No. WO 98/07697 (published Feb. 26,1998), PCT Publication No WO 98/03516 (published Jan. 29, 1998), PCTPublication No WO 98/34918 (published Aug. 13, 1998), PCT Publication NoWO 98/34915 (published Aug. 13, 1998), PCT Publication No WO 98/33768(published Aug. 6, 1998), PCT Publication No WO 98/30566 (published Jul.16, 1998), European Patent Publication 606,046 (published Jul. 13,1994), European Patent Publication 931,788 (published Jul. 28, 1999),PCT Publication No WO 90/05719 (published May 31, 1990), PCT PublicationNo WO 99/52910 (published Oct. 21, 1999), PCT Publication No WO 99/52889(published Oct. 21, 1999), PCT Publication No WO 99/29667 (publishedJun. 17, 1999), PCT International Application No. PCT/IB98/01113 (filedJul. 21, 1998), European Patent Application No. 99302232.1 (filed Mar.25, 1999), U.S. Pat. No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No.5,861,510 (issued Jan. 19, 1999), and European Patent Publication780,386 (published Jun. 25, 1997). In one example, the MMP inhibitors donot induce arthralgia upon administration. In another example, the MMPinhibitor selectively inhibits MMP-2 and/or MMP-9 relative to the othermatrix-metalloproteinases (such as MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specificexamples of MMP inhibitors of use are AG-3340, RO 32-3555, RS 13-0830,3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxaicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofsaid compounds.

The antibodies that specifically bind IGF-I and IGF-II with highaffinity, such as at least picomolar affinity, antigen binding fragmentthereof, bispecific form thereof, can also be used with signaltransduction inhibitors, such as agents that can inhibit EGF-R(epidermal growth factor receptor) responses, such as EGF-R antibodies,EGF antibodies, and molecules that are EGF-R inhibitors; VEGF (vascularendothelial growth factor) inhibitors, such as VEGF receptors andmolecules that can inhibit VEGF; and erbB2 receptor inhibitors, such asorganic molecules or antibodies that bind to the erbB2 receptor, forexample, HERCEPTIN™ (Genentech, Inc.). EGF-R inhibitors are describedin, for example in PCT Publication Nos. WO 95/19970 (published Jul. 27,1995), WO 98/14451 (published Apr. 9, 1998), WO 98/02434 (published Jan.22, 1998), and U.S. Pat. No. 5,747,498 (issued May 5, 1998).EGFR-inhibiting agents also include, but are not limited to, themonoclonal antibodies C225 and anti-EGFR 22Mab (ImClone SystemsIncorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA),EMD-5590 (Merck KgaA), MDX-447/H-477 (Medarex Inc. and Merck KgaA), andthe compounds ZD-1834, ZD-1838 and ZD-1839 (AstraZeneca), PKI-166(Novartis), PKI-166/CGP-75166 (Novartis), PTK 787 (Novartis), CP 701(Cephalon), leflunomide (Pharmacia/Sugen), CI-1033 (Warner Lambert ParkeDavis), CI-1033/PD 183,805 (Warner Lambert Parke Davis), CL-387,785(Wyeth-Ayerst), BBR-1611 (Boehringer Mannheim GmbH/Roche), Naamidine A(Bristol Myers Squibb), RC-394011 (Pharmacia), BIBX-1382 (BoehringerIngelheim), OLX-103 (Merck & Co.), VRCTC-310 (Ventech Research), EGFfusion toxin (Seragen Inc.), DAB-389 (Seragen/Lilgand), ZM-252808(Imperial Cancer Research Fund), RG-50864 (INSERM), LFM-A12 (ParkerHughes Cancer Center), WH1-P97 (Parker Hughes Cancer Center), GW-282974(Glaxo), KT-8391 (Kyowa Hakko) and EGF-R Vaccine (York Medical/Centro deImmunologia Molecular (CIM)).

VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.), SH-268(Schering), and NX-1838 (NeXstar) can also be used in conjunction withan antibody that specifically binds IGF-II. VEGF inhibitors aredescribed in, for example in PCT Publication No. WO 99/24440 (publishedMay 20, 1999), PCT International Application PCT/IB99/00797 (filed May3, 1999), PCT Publication No. WO 95/21613 (published Aug. 17, 1995), PCTPublication No. WO 99/61422 (published Dec. 2, 1999), U.S. Pat. No.5,834,504 (issued Nov. 10, 1998), PCT Publication No. WO 98/50356(published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar. 16,1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No.5,792,783 (issued Aug. 11, 1998), PCT Publication No. WO 99/10349(published Mar. 4, 1999), PCT Publication No. WO 97/32856 (publishedSep. 12, 1997), PCT Publication No. WO 97/22596 (published Jun. 26,1997), PCT Publication No. WO 98/54093 (published Dec. 3, 1998), PCTPublication No. WO 98/02438 (published Jan. 22, 1998), WO 99/16755(published Apr. 8, 1999), and PCT Publication No. WO 98/02437 (publishedJan. 22, 1998). Other examples of some specific VEGF inhibitors areIM862 (Cytran Inc.); anti-VEGF monoclonal antibody of Genentech, Inc.;and angiozyme, a synthetic ribozyme from Ribozyme and Chiron. These andother VEGF inhibitors can be used in conjunction with an antibody thatspecifically binds IGF-II.

ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome pic), andthe monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc.) and 2B-1(Chiron), can furthermore be combined with the compound of theinvention, for example those indicated in PCT Publication No. WO98/02434 (published Jan. 22, 1998), PCT Publication No. WO 99/35146(published Jul. 15, 1999), PCT Publication No. WO 99/35132 (publishedJul. 15, 1999), PCT Publication No. WO 98/02437 (published Jan. 22,1998), PCT Publication No. WO 97/13760 (published Apr. 17, 1997), PCTPublication No. WO 95/19970 (published Jul. 27, 1995), U.S. Pat. No.5,587,458 (issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305 (issuedMar. 2, 1999). ErbB2 receptor inhibitors of use are also described inU.S. Provisional Application No. 60/117,341, filed Jan. 27, 1999, and inU.S. Provisional Application No. 60/117,346, filed Jan. 27, 1999.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of at least one of the antibodies disclosed herein toeffectively treat the patient. The dosage can be administered once butmay be applied periodically until either a therapeutic result isachieved or until side effects warrant discontinuation of therapy. Inone example, a dose of the antibody is infused for thirty minutes everyother day. In this example, about one to about ten doses can beadministered, such as three or six doses can be administered every otherday. In a further example, a continuous infusion is administered forabout five to about ten days. The subject can be treated at regularintervals, such as monthly, until a desired therapeutic result isachieved. Generally, the dose is sufficient to treat or amelioratesymptoms or signs of disease without producing unacceptable toxicity tothe patient. In one example, the dose is sufficient to decrease thephosphorylation of the IGF-I receptor.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein, such as a cytotoxin or a drug, as a central core.In microspheres the therapeutic is dispersed throughout the particle.Particles, microspheres, and microcapsules smaller than about 1 μm aregenerally referred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992) both of which are incorporated herein by reference.

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496).

Diagnostic Methods and Kits

A method is provided herein for the detection of the expression of IGF-Iand/or IGF-II in vitro or in vivo. In one example, expression of IGF-Iand/or IGF-II is detected in a biological sample. The sample can be anysample, including, but not limited to, tissue from biopsies, autopsiesand pathology specimens. Biological samples also include sections oftissues, for example, frozen sections taken for histological purposes.Biological samples further include body fluids, such as blood, serum,plasma, sputum, spinal fluid or urine. A biological sample is typicallyobtained from a mammal, such as a rat, mouse, cow, dog, guinea pig,rabbit, or primate. In one embodiment, the primate is macaque,chimpanzee, or a human.

In several embodiments, a method is provided for detecting a malignancysuch as, but not limited to, a sarcoma, leukemia, prostate cancer, lungcancer, breast cancer, colon cancer, stomach cancer, uterine cancer,cervical cancer, esophageal cancer, liver cancer, pancreatic cancer,kidney cancer, thyroid cancer, brain cancer, or an ovarian cancer.

In additional embodiments, a method is provided for detecting multiplemyeloma, liquid tumor, liver cancer, thymus disorder, T-cell mediatedauto-immune disease, endocronological disorder, ischemia,neurodegenerative disorder, lung cancer, bone cancer, pancreatic cancer,skin cancer, cancer of the head and neck, cutaneous or intraocularmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, colon cancer, breast cancer, gynecologictumors (such as uterine sarcomas, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina or carcinoma of the vulva), Hodgkin's disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrine system(such as cancer of the thyroid, parathyroid or adrenal glands), sarcomasof soft tissues, cancer of the urethra, cancer of the penis, prostatecancer, chronic or acute leukemia, solid tumors of childhood,lymphocytic lymphomas, cancer of the bladder, cancer of the kidney orureter (such as renal cell carcinoma, carcinoma of the renal pelvis), orneoplasms of the central nervous system (such as primary CNS lymphoma,spinal axis tumors, brain stem gliomas or pituitary adenomas). A methodis also provided for determining the prognosis of a subject with any ofthe malignancies listed above.

Methods are provided for detecting IGF-I and/or IGF-II in a biologicalsample, wherein the method includes contacting a biological sample witha human antibody that binds IGF-I and IGF-II with picomolar affinity,antigen binding fragment thereof, or bispecific form thereof, underconditions conductive to the formation of an immune complex, anddetecting the immune complex, to detect the IGF-I and/or IGF-II in thebiological sample. In one example, the detection of IGF-I and/or IGF-IIin the sample indicates that the subject has a malignancy. In anotherexample, the detection of IGF-I and/or IGF-II in the sample indicatesthat the subject is prone to metastasis.

In one embodiment, the human antibody that specifically binds IGF-I andIGF-II with high affinity, such as at least picomolar affinity, antigenbinding fragment or bispecific form is directly labeled with adetectable label. In another embodiment, the human antibody thatspecifically binds IGF-I and IGF-II with high affinity, such a picomolaraffinity (the first antibody, antigen binding fragment thereof, orbispecific form thereof, is unlabeled and a second antibody or othermolecule that can bind the human antibody that specifically binds IGF-Iand IGF-II (or the antigen binding fragment or bispecific form) islabeled. As is well known to one of skill in the art, a second antibodyis chosen that is able to specifically bind the specific species andclass of the first antibody. For example, if the first antibody is ahuman IgG, then the secondary antibody may be an anti-human-IgG. Othermolecules that can bind to antibodies include, without limitation,Protein A and Protein G, both of which are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, IGF-I and/or IGF-II can be assayed in abiological sample by a competition immunoassay utilizing IGF-I and/orIGF-II standards labeled with a detectable substance and an unlabeledhuman antibody that specifically binds IGF-I and IGF-II with highaffinity, such as picomolar affinity, an antigen binding fragmentthereof or a bispecific form thereof. In this assay, the biologicalsample, the labeled IGF-I and/or IGF-II standards and the human antibodythat specifically bind IGFI and IGF-II (or the antigen binding fragmentor bispecific form) are combined and the amount of labeled IGF-I and/orIGF-II standard bound to the unlabeled antibody is determined. Theamount of IGF-I and/or IGF-II in the biological sample is inverselyproportional to the amount of labeled IGF-I and/or IGF-II standard boundto the antibody that specifically binds IGF-I and IGF-II (or bound tothe antigen binding fragment or bispecific form).

The immunoassays and method disclosed herein can be used for a number ofpurposes. In one embodiment, the human antibody that specifically bindsIGF-I and IGF-II, antigen binding fragment, or bispecific form, can beused to detect the production of IGF-I and/or IGF-II in cells in cellculture. In another embodiment, the antibody can be used to detect theamount of IGF-I and/or IGF-II in a biological sample.

Increased expression of IGF is associated with several types of cancer,including a sarcoma, leukemia, prostate cancer, lung cancer, breastcancer, colon cancer, stomach cancer, uterine cancer, cervical cancer,esophageal cancer, liver cancer, pancreatic cancer, kidney cancer,thyroid cancer, brain cancer, or an ovarian cancer. Thus, the level ofIGFs can be used to diagnose, or determine the prognosis of, a sarcoma,leukemia, prostate cancer, lung cancer, breast cancer, colon cancer,stomach cancer, uterine cancer, cervical cancer, esophageal cancer,liver cancer, pancreatic cancer, kidney cancer, thyroid cancer, braincancer, or an ovarian cancer, in a subject.

In one embodiment, a kit is provided for detecting IGF-I and IGF-II in abiological sample, such as a blood sample. Kits for detecting apolypeptide will typically comprise a human antibody that specificallybinds IGF-I and IGF-II, such as any of the antibodies disclosed herein,including the antigen binding fragments and bispecific forms. In someembodiments, an antibody fragment, such as an Fv fragment is included inthe kit. For in vivo uses, the antibody can be a scFv fragment. In afurther embodiment, the antibody is labeled (for example, with afluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds IGF-I and IGF-II (orthe antigen binding fragment or bispecific form thereof). Theinstructional materials may be written, in an electronic form (such as acomputer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting IGF-I and/or IGF-II in a biologicalsample generally includes the steps of contacting the biological samplewith an antibody, antigen binding fragment or bispecific antibody whichspecifically reacts, under immunologically reactive conditions, to IGF-Iand IGF-II polypeptide. The antibody is allowed to specifically bindunder immunologically reactive conditions to form an immune complex, andthe presence of the immune complex (bound antibody) is detected directlyor indirectly.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies, antigen bindingfragments, and bispecific forms can be conjugated to other compoundsincluding, but not limited to, enzymes, magnetic beads, colloidalmagnetic beads, haptens, fluorochromes, metal compounds, radioactivecompounds or drugs. The antibodies can also be utilized in immunoassayssuch as but not limited to radioimmunoassays (RIAs), enzyme linkedimmunosorbant assays (ELISA), or immunohistochemical assays. Theantibodies can also be used for fluorescence activated cell sorting(FACS). A FACS employs a plurality of color channels, low angle andobtuse light-scattering detection channels, and impedance channels,among other more sophisticated levels of detection, to separate or sortcells (see U.S. Pat. No. 5,061,620). Any of the human antibodies thatspecifically bind IGF-II, antigen binding fragments and bispecificforms, as disclosed herein, can be used in these assays. Thus, theantibodies can be used in a conventional immunoassay, including, withoutlimitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Westernblot or immunoprecipitation.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

The isolation, maturation and characterization of a phage-derived humanmonoclonal antibody (m708.5) which bound with very high affinity toIGF-I (K_(D)=200 pM) and IGF-II (K_(D)=60 pM) is disclosed herein. Thisantibody potently inhibited signal transduction mediated by the IGF-IRinteraction with both IGF-I and IGF-II, resulting in the inhibition ofphosphorylation of IGF-IR and cancer cell growth.

Example 1 Materials and Methods

Phage Display Fab Library Panning:

Recombinant human IGF-I was used to screen a human naive Fab phagelibrary containing 10¹⁰ unique clones using protocols and reagentssimilar to those that were used previously (Zhu and Dimitrov, MethodsMol Biol, 2009. 525: p. 129-42). Recombinant human IGF-I was conjugatedonto magnetic beads (Invitrogen) as target for the library panning. The10 μg of the antigen was used in the first round of panning. The 10¹²amplified phages were used for the panning. After 10 washes, boundphages on the beads were directly used to infect exponentially growingTG1 cells and rescued by M13KO7 helper phage. Panning was repeated tworounds by using 2 μg of antigen each round. Two hundred individualcolonies after the third round were picked and inoculated into 2YTmedium in 96-well plate for phage ELISA screening.

Generation and Selection of the Light Chain-shuffled Phage DisplayLibrary: The original human Fab phage display library was used as thesource of the V_(L) repertoire in the shuffled library. The phagemidpreparation from the original library was first digested withrestriction enzymes NcoI and SpeI and followed by electrophoresis on anagarose gel to delete the entire V_(H) repertoire. The gene encoding theVH domain of clone m708 was amplified by error-prone PCR kit fromStratagene to introduce random mutations and then fused with CH₁ genefragment by SOE PCR. The fused fragment was digested with NcoI and SpeI, purified from agarose gel and then ligated into the purified backbonevector to create the V_(L)-shuffled Fab repertoire. E. coli TG1 cellswere transformed with the ligation mixtures by electroporation. Thetransformed TG1 cells were plated on 2YT agar plates containing 100μg/ml ampicillin and 2% glucose. After incubation overnight at 37° C.,all of the colonies grown on the plates were scraped into 5 ml of 2YTmedium containing 2% glucose and 100 μg/ml ampicillin, mixed with 1.2 mlof 50% glycerol (final concentration 10%), aliquoted, and stored at −70°C. as the mutant library stock.

The library stock was grown to log phase in 20 ml of 2YT medium, rescuedwith M13K07 helper phage, and amplified overnight in 2YT mediumcontaining 100 μg/ml of ampicillin and 50 μg/ml of kanamycin at 30° C.The phage preparation was precipitated in 4% PEG8000 with 0.5 M NaCl anddissolved in 1 ml of PBS as phage library stock. Two rounds ofbiopanning were performed on human IGF-I conjugated magnetic beads asdescribed in the original library panning.

Mutagenesis by Error-Prone PCR:

Error-prone PCR of the entire scFv gene was performed using STRATAGENEGENEMORPH® II Random Mutagenesis Kit according to the instructions ofthe manufacturer. Briefly, PCR was done in a 50-μL reaction containing1× Mutazyme II reaction buffer, 0.5 μM each of primers

RDlinker1F (SEQ ID NO: 15) 5′ GATATATCCATGGCCCAGGCGGCC 3′ and ERRORR(SEQ ID NO: 16) (5′ ACCACTAGTTGGGCCGGCCTG 3′),0.2 mM (each) dNTPs, 1 ng of DNA template, 2 μM 8-oxo-deoxyguanosinetriphosphate, 2 μM 2′-deoxy-p-nucleoside-5′-triphosphate, and 2.5 U ofMutazyme II DNA polymerase. The reaction mixtures were denatured at 95°C. for 2 min, cycled 35 times at 95° C. for 1 min, 60° C. for 1 min, and72° C. for 1 min, and finally extended at 72° C. for 10 min. The PCRproducts were purified by 1% agarose gel electrophoresis and eachamplified in four 100-1 μL PCR reactions containing 1× Accuprime PCRreaction mix (Invitrogen), 1 μM of primers

YDRDF (SEQ ID NO: 17) (5′ CTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTGGCCCAGGCGGCC 3′) and YDRDR (SEQ ID NO: 18) (5′GAGCCGCCACCCTCAGAACCGCCACCCTCAGAGCCACCACTAGTT GGGCCGGCCTG 3′),120 ng of error-prone PCR product, and 2.5 U of Accuprime pfx DNApolymerase (Invitrogen). The reactions were thermally cycled at the sameconditions except that 30 cycles were used. Reaction products werepurified by 1% agarose gel electrophoresis and concentrated withultrafilter in water.

Construction of Yeast Displayed Mutant Library:

The plasmid used for library construction, pYD7, was modified frompCTCON2 (Hackel et al., J Mol Biol, 2008. 381(5): p. 1238-52; Chao, G etal. Nat Protoc, 1(2): 755-768). Two SfiI restriction sites of pYD7before and after the scFv matched the cloning sites of the pComb3Xvector. This allowed fragments to be shuttled between the yeast vectorand pComb3x. To avoid interference of agglutinin protein aga2p, it wasalso converted to 3′ end of antibody fragment.

The vector was digested with SfiI. Multiple aliquots of 24 μg ofmutagenized scFv gene and 8 μg of plasmid vector were combined with20-30 μL of water. The pYD7-scFv libraries were then transformed intoEBY100 using electroporation transformation method (Benatuil et al.,Protein Eng Des Sel, 2010. 23(4): p. 155-9). Homologous recombination ofthe linearized vector and degenerated insert DNA yielded intact plasmid(Hackel, supra). Briefly, the 10 ml of EBY100 yeast cells in YPD media(10 g/l yeast nitrogen base, 20 g/l peptone and 20 g/l glucose) weregrown overnight. The culture was inoculated into fresh 100 ml of YPDmedia to reach OD₆₀₀ of 1.6 before collecting by centrifugation. Thecell pellet was washed twice with 50 ml of cold water, and once with 50ml cold electroporation buffer (1 M sorbitol/1 mM CaCl₂). Cells wereconditioned in 20 ml of 0.1 M LiAc/10 mM DTT by incubation in culture at30° C. for 30 minutes, washed one more time with 50 ml ofelectroporation buffer, then resuspended in the same buffer to reach 1ml volume. Each electroporation mixture included 400 μl of yeast cellsuspension, 4 μg linearized vector, and 12 μg insert DNA. Cells wereelectroporated at 2.5 kV and 25 mF in BioRad GenePulser cuvettes (0.2 cmelectrode gap). After electroporation, cells were resuspended in 20 mlof 1:1 (y:y) mix of 1 M sorbitol. YPD media and incubated at 30° C. for1 h. Finally, the cells were collected and cultured in SDCAA media (20g/l glucose, 6.7 g/l yeast nitrogen base without amino acids, 5.4 g/lNa₂HPO₄, 8.6 g/l NaH₂PO₄.H₂O and 5 g/l casamino acids) at 30° C. with250 rpm shaking for 1-2 days.

Selection of Binders from the Yeast Libraries:

The yeast libraries were grown as previously described (Chao et al., NatProtoc, 2006. 1(2): p. 755-68). Typically yeast were grown in SDCAAmedia for 18 h at 30° C. and then transferred to SG/RCAA media (20 g/lgalactose, 20 g/l Raffinose, 1 g/l glucose, 6.7 g/l yeast nitrogen basewithout amino acids, 5.4 g/l Na₂HPO₄, 8.6 g/l NaH₂PO₄.H₂O and 5 g/lcasamino acids) for 16-18 h at 20° C. in culture volumes appropriate forthe size of the library.

The methodology for generating and isolating higher affinity mutants wasas described (Chao et al., supra; Boder et al., Proc Natl Acad Sci USA,2000. 97(20): p. 10701-5; Boder et al., Methods Enzymol, 2000. 328: p.430-44). Antigen concentrations are chosen based on the expecteddissociation constant (K_(D)) of the parental. The antigen incubationvolume must be large enough to allow at least antigen excess of tenfoldover amounts of scFv displayed on yeast. Antigen incubation times werebased to come to equilibrium calculated as the reference(Garcia-Rodriguez, et al., Nat Biotechnol, 2007. 25(1): p. 107-16).Before FACS selection, yeast cells (1×10⁹) were incubated with 10μg-IGF-I conjugated magnetic beads for 1 h at room temperature in PBSAbuffer (0.1% BSA in PBS), followed by separation with magnetic stand.The isolated beads were washed 3 times with PBSA buffer, put into 10 mlof SDCAA media and grown overnight in a 30° C. shaker with 250 rpm. Theyeast cells recovered from magnetic beads were induced in SG/RCAA mediafor 18 h at 20° C. with 250 rpm shaking. For 1^(st) round (3 FACSselection), the first selection, approximately 1×10⁸ yeast cells werepelleted, washed twice with PBSA buffer and resuspended in 1 ml PBSAbuffer with 3 nM biotinylated IGF-I and a 1:100 dilution of mouseanti-c-myc antibody (Invitrogen). After incubation, yeast cells werewashed 3 times and then resuspended in 1 ml PBSA buffer. Both 1:50dilution of R-phycoerythrin conjugated Streptavidin (Invitrogen) andAlexa488 conjugated goat anti-mouse IgG antibody (Invitrogen) was addedto yeast cells, incubated at 4° C. for 30 min, and washed 3 times withPBSA buffer again, and then resuspended in PBSA buffer for flowcytometric sorting. Sort gates were determined to select only populationwith higher antigen binding signals. Collected cells were grownovernight in SDCAA media at 30° C. and induced in SG/RCAA for the nextround of sorting. For the next two selections, approximately 1-2×10⁷yeast cells were used for staining with 1 nM and 0.3 nM biotinylatedIGF-I, respectively.

Two and three selections were performed in the 2^(nd) and 3^(rd) round.Yeast cells were pelleted, washed in PBSA buffer, resuspended in PBSAbuffer with biotinylated IGF-I (ranging from 3 nM to 0.1 nM) and mouseanti-cmyc antibody, and incubated on ice. Cells were then washed withPBSA twice and resuspended in 1 ml PBSA with R-phycoerythrin conjugatedStreptavidin and Alexa488 conjugated goat anti-mouse antibody.

After the 1^(st) and 2^(nd) round, yeast plasmids were isolated usingZYMOPREP™ Yeast Plasmid Miniprep II kit (Zymo Research) according to themanufacturer's instructions and used for templates of libraryconstruction. Cells from round 3 spread on SDCAA plates. Plasmids fromthe 3^(rd) round were prepared, sequenced and characterized.

Conversion to IgG1:

Fab and scFv were cloned into pDR12, which allows simultaneousexpression of the heavy chain and light chains. Briefly, the heavy chainvariable region was first cloned into pDR12 via XbaI and SacI sites. Thelight chain sequence (VL+CL) was then cloned into pDR12 via HindIII andEcoRI sites.

Expression of Fab, scFv and IgG1:

Fab and scFv were expressed and purified as previously described (Zhu etal., Proc Natl Acad Sci USA, 2007. 104(29): p. 12123-8; Zhao et al.,Protein Expr Purif, 2009. 68(2): p. 190-5). HB2151 cells weretransformed with pComb3x plasmid containing Fab or scFv sequences.Single fresh colonies were inoculated into 2YT medium containing 100μg/mL ampicillin and 0.2% glucose. The culture was grown at 37° C. with250 rpm until A₆₀₀ reached 0.5. Isopropyl-L-thio-h-D-galactopyranoside(final concentration 0.5 mM) was added to induce expression. Afterovernight growth at 30° C., the bacteria were centrifuged at 5,000×g for15 minutes. Bacteria were centrifuged at 5,000×g for 15 minutes. Thepellet was resuspended in PBS with polymyxin B (10,000 units/mL).Soluble Fab was released from periplasm by incubating at 30° C. for 30minutes. The extract was clarified at 15,000×g for 30 minutes. The clearsupernatant was recovered for purification on Ni-NTA column. RecombinantFabs have FLAG™ and His tags.

IgGs were expressed in 293 FREESTYLE™ cells. CELLFECTIN® was used totransfect 293 FREESTYLE™ cells according to the instructions of themanufacturer (Invitrogen). Four days posttransfection, the culturesupernatant was harvested. IgGs were purified on protein G column.

ELISA Binding Assay:

Antigens (50 ng) per well were coated on 96-well ELISA plates overnightat 4° C. For phage ELISA, ˜1×10¹⁰ phages were incubated with antigen for1 h. Bound phage was detected with anti-M13-HRP polyclonal antibody(Pharmacia, Piscataway, N.J.). For Fab and IgG ELISA, Fabs and IgGs withdifferent dilutions were incubated with antigens for 1 hour. Bound Fabswere detected with anti-FLAG-HRP mAb (1:3,000; Sigma). Bound IgGs weredetected with anti-human Fc-HRP mAb (1:2,000; Invitrogen). The2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) substrate wasadded and the reaction was read at 450 nm.

Affinity Measurement of Yeast-Displayed scFv:

The equilibrium dissociation constant for a clone was determinedessentially as described (Chao et al, Nat Protoc, 2006. 1(2): p.755-68). Briefly, the deserved yeast clone was grown and induced as forFACS analysis. The 1×10⁵ yeast cells were incubated with biotinylatedIGF-I at different concentrations and a 1:100 dilution of mouseanti-c-myc antibody for 3 h at room temperature in PBSA buffer. Six toeight different concentrations of biotinylated antigens were chosenaround the equilibrium dissociation constant. Incubation volumes andnumber of yeast stained were chosen to keep the number of antigenmolecules in tenfold excess above the number of scFv. Cells were thenwashed once in PBSA buffer. The cells were stained with a 1:50 dilutionof R-phycoerythrin conjugated Streptavidin and Alexa488 conjugated goatanti-mouse antibody on ice in the dark, then washed again andresuspended in 0.5 ml wash buffer. Analysis was performed using a BDBioscience FACS.

Affinity Determination by Surface Plasmon Resonance:

Interactions between various isolated antibodies and IGF-I and IGF-IIwere analyzed by surface plasmon resonance technology using a BiacoreX100 instrument (GE healthcare). IGF-I or IGF-II was covalentlyimmobilized onto a sensor chip (CM5) using carbodiimide couplingchemistry. A control reference surface was prepared for nonspecificbinding and refractive index changes. For analysis of the kinetics ofinteractions, varying concentrations of antibodies were injected at flowrate of 30 μl/min using running buffer containing 150 mM NaCl, 3 mMEDTA, and 0.005% P-20 (pH 7.4). The association and dissociation phasedata were fitted simultaneously to a 1:1 Langumir global model by usingthe nonlinear data analysis program BIAevaluation 3.2. All theexperiments were done at 25° C.

Competition Assay:

IgG m708.5 was incubated with 5 nM biotinylated IGF-I and biotinylated 1nM IGF-II at room temperature for 20 min, respectively. Then, themixtures were added to 5×10⁵ MCF-7 cells in 50 μl PBSA buffer andincubated for 30 min on ice. After one washing, cells were incubatedwith a 1:50 dilution of R-phycoerythrin conjugated Streptavidin for 30min on ice, then washed again and resuspended in 0.5 ml PBSA buffer.Analysis was performed using a BD Bioscience FACS. Irrelevantanti-gp41MPER scFv-3A2a and anti-Nipah/Hendra viruses IgG-m102.4 (Zhu etal., J Infect Dis, 2008. 197(6): p. 846-53) were chosen as negativecontrols.

Phosphorylation Assay:

MCF-7 cells were seeded in a six-well plate at 1×10⁶ cells per well inthe complete growth medium (DMEM with 10% FBS, 100 U/ml penicillin, 100μg/ml streptomycin). After overnight culture, cells were rinsed withserum-free DMEM and then cultured in serum-free DMEM for 5 hours. Thetreatment medium was made by adding 1 nM IGF-I or 5 nM IGF-II andvarious concentrations of IgG in serum-free DMEM. After pre-incubationfor 30 min at RT, the treatment medium was added to cells. Twentyminutes after addition of ligands and antibodies, cells were chilled onice, rinsed in cold PBS, and lysed in 1 mL of lysis buffer [50 mmol/LHEPES (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5mmol/L MgCl2, 2 mmol/L sodium vanadate, and protease inhibitors].Lysates were kept on ice for 30 minutes, followed by centrifugation at17,000×g for 30 minutes. The supernatant was used forimmunoprecipitation: 20 μL of protein G Sepharose 4B and 3 μg of rabbitanti-IGFIR β IgG (C-20, Santa Cruz) at 4° C. overnight. After extensivewash, the immunoprecipitates were run on 4% to 12% NUPAGE, transferredto polyvinylidene difluoride membrane, and blotted withanti-phosphotyrosine mAb PY20 (Sigma). The membrane was stripped andreprobed with C-20 polyclonal antibody to detect total IGFIR in theimmunoprecipitates. A similar procedure was used to detect thephosphorylation of insulin receptor but immunoprecipitation and Westernblots were done with rabbit anti-IR β pAb (C-19, Santa Cruz) which isspecific to insulin receptor.

Cell Growth Assay:

MCF-7 cells were seeded in a 96-well plate at 1×10⁴ cells per well incomplete growth medium. After overnight culture, cells were rinsed withserum-free DMEM. Various concentrations of IgGs were mixed with IGF-I(2.5 nM) and IGF-II (2.5 nM) and incubated for 20 min at roomtemperature. Then, 100 μl of the mixture was added each wellimmediately. Cells were allowed to grow for 3 days, and 20 μl of MTSsubstrate (Promega) was added to detect viable cells. Plates wereincubated at 37° C. for 1 h and monitored at A₄₅₀ nm. Cells inserum-free medium with IGF ligands were as positive control. Cells inserum-free medium without IGF ligands were as blank control.

Example 2 Identification of m708.2 Cross-Reactive IGF-I and IGF-II

To develop human mAbs that specifically bind IGF-I, a large (size ˜10¹⁰)naive human Fab phage-displayed library was utilized. Recombinant humanIGF-I was conjugated to magnetic beads and used as an antigen forpanning. After three rounds of panning, more than 200 random individualphage clones were screened by phage ELISA against IGF-I. Clones thatexhibited significant binding to IGF-I were sequenced. Finally, threeclones with unique sequences were found. They were expressed in bacteriaas soluble Fabs, purified, and tested for binding activity in ELISA. TwoFabs, designated m705 and m706 showed binding specifically to IGF Ionly, while one Fab, m708, exhibited significant levels of binding toboth IGF-I and IGF-II in ELISA (FIG. 1). Thus, m708 was selected foraffinity maturation by light chain shuffling.

Two rounds of panning of the light chain shuffled library (containing2×10⁸ independent clones) against IGF-I conjugated magnetic bead wereperformed and 200 clones from the second round of panning were screenedby phage ELISA. Of these 200 clones, only m708.2 showed markedly higherbinding to both IGF-I and IGF-II than parental m708 version.

Example 3 Construction of Libraries of m708.2 Mutants Displayed on Yeast

The binding affinity of m708.2 was further improved by utilizing yeastdisplay technique. The scFv gene constructed from m708.2 was randomlymutagenized by employing error-prone PCR strategy. The mutant librarywas incorporated into a yeast display system by homologous recombinationwith a vector containing C-terminal Aga2 protein and c-myc tag. Usually,library transformation yielded 5×10⁷ clones per microgram vector DNA.High mutation frequencies (˜1%) can also be achieved by using 1 ng oftarget DNA with 35 PCR cycles. In order to obtain large amounts of DNAinsert and avoid improperly incorporation at the site of homologousrecombination, a small purified DNA of the first PCR reaction wasre-amplified in a second PCR reaction. Thus, 30-40% of the transformedcells displayed scFvs verified by flow cytometry analysis.

Example 4 Selection of an Affinity Matured scFv (m708.5)

Yeast libraries of relatively large (up to 10⁹) size were generated andtherefore 10⁹-10¹⁰ cells should be sorted (Blaise et al., Gene, 2004.342(2): p. 211-8). Magnetic separation methods are capable of processinglarge numbers of cells. Before FACS sorting, the mutagenesis yeastlibraries were subjected to one round of selection by using IGF-Iconjugated magnetic beads (Yeung et al., Biotechnol Prog, 2002. 18(2):p. 212-20). This allowed elimination of non-expression and weak bindingyeast cells from the libraries. The affinity maturation scheme is shownin FIG. 2. Briefly, a yeast library of m708.2 mutants was constructed.The library was screened on IGF-I conjugated magnetic beads once andsorted several times by FACS for binding to IGF-I. The sorted populationwas mutated by error-prone PCR of the entire gene to yield a newsub-library. The process of sorting and mutagenesis was then cyclicallyrepeated. The highest affinity clones from the final round of maturationwere identified and their sequences analyzed.

One dominant clone, m708.5, was identified by sequence analysis. Whencompared with m708.2, m708.5 had two amino acid substitutions in CRD-H2,two amino acid substitutions in CDR-H3 and nine amino acid substitutionsin the framework region (Table 1).

TABLE 1  Sequences of cross-reactive antibodies  to IGF-I and IGF-IIClone H1 H2 H3 m708.2 GGTFSSYA IIPILGIA ARGPRGYSYNFDY m708.5 -----------T---V -G----------N Antigen-binding loops H1, H2 and H3 (VH) indicatethe loop residues with CDRs that were subjected to mutation. For theH-CDR sequences of m708.5, see amino acids 26-33 of SEQ ID NO: 7 (H1),amino acids 51-58 of SEQ ID NO: 7 (H2), and amino acids 97-109 of SEQ IDNO: 7 (H3). For the H-CDR sequences of m708.2, see amino acids 26-33 ofSEQ ID NO: 9 (H1), amino acids 51-58 of SEQ ID NO: 9 (H2), amino acids97-109 of SEQ ID NO: 9 (H3).

These substitutions resulted in a remarkable improvement of affinity,while the antibody surprisingly still retained cross-reactivity. Theaffinities of m708.2 and m708.5 were calculated by incubating eachyeast-displayed scFv with varying concentrations of biotinylated IGF-Ior IGF-II. The affinity of yeast-displayed m708.5 scFv for IGF-Iimproved 39-fold compared to m708.2 (1×10⁻¹⁰ M versus 3.9×10⁻⁹ M,respectively), whereas its affinity for IGF-II increased 27-fold, from1.1×10⁻⁹ M to 4.1×10⁻¹¹ M (Table 2). Similar values were obtained forisolated II, compared to m708.2 scFv (2.2×10⁻⁹ M for IGF-1 and 1.8×10⁻⁹Mfor IGF-II) (Table 2).

TABLE 2 Affinity and binding kinetics of cross- reactive antibodies toIGF-I and IGF-II Anti- Anti- FACS Biacore body gen K_(D) (M⁻¹) k_(on)(M⁻¹s⁻¹) k_(off) (s⁻¹) K_(D) (M⁻¹) m708.2 IGF-I 3.9 × 10⁻⁹ 1.1 × 10⁶ 2.4× 10⁻³ 2.2 × 10⁻⁹ scFv IGF-II 1.1 × 10⁻⁹ 2.3 × 10⁵ 4.0 × 10⁻⁴ 1.8 × 10⁻⁹m708.5 IGF-I 1.0 × 10⁻¹⁰ 1.4 × 10⁶ 2.8 × 10⁻⁴ 2.0 × 10⁻¹⁰ scFv IGF-II4.1 × 10⁻¹¹ 4.1 × 10⁶ 2.5 × 10⁻⁶ 6.1 × 10⁻¹¹

Therefore, the cross-reactive antibody m708.2 was successfully maturedto picomolar affinities for both IGF-I and IGF-II.

Example 5 Avidity of IgG1 m708.5

To determine whether changes in affinity for scFv were observed in IgGformat, the scFv was converted to IgG. As with scFv, m708.5 IgG showedincreased affinity for IGF-I and IGF-II. The effective K_(D) of m708.5for IGF-I and IGF-II was 1000-fold and 60-fold higher than the K_(D) forthe scFv, respectively. The measured K_(D) (<10⁻¹² M) is beyond thesensitive limitation of Biacore instrument (10⁶ M⁻¹ S⁻¹ for k_(on) and10⁻⁶ S⁻¹ for k_(off)). IgG1 m708.5 was also much more effective inbinding to both IGF-I and IGF-II than m708.2 as measured by ELISA (FIG.4). Thus, m708.5 retains its high binding affinity in IgG1 format and inthe scFv format.

Example 6 Blocking of IGF-I and IGF-II Bound to MCF-7 Cells

MCF-7 express both IGF-IR and IR. A competitive assay was performed todetermine whether m708.5 blocked the binding of IGF-I and IGF-II toIGF-IR and IR on MCF-7 breast cancer cells by flow cytometry. M708.5 waspre-incubated with IGF-I and IGF-II, respectively. The antibodies wereallowed to bind to MCF-7 cells. Irrelevant 3A2a scFv and m102.4 IgG asnegative control were also mixed with IGF ligands at the same condition.M708.5 in scFv and IgG1 formats completely blocked IGF-I and IGF-IIbinding to their receptors on MCF-7 cells (FIG. 5). Control scFv and IgGthat do not recognize IGFs had no effect. Thus, m708.5 could blockIGF-I/II-mediated signals inducing the activation and phosphorylation ofIGF-IR and IR on cancer cells.

Example 7 Inhibition of IGF-I and IGF-II Induced IGF-IR Phosphorylation

The antibody also inhibited IGF-I-induced (1 nM) or IGF-II-induced (5nM) phosphorylation. Immunoblottings (FIG. 6 a) showed that from 1 nM to100 nM, m708.5 completely inhibited the IGF-I-induced phosphorylation ofIGFIR. Similar inhibitory activity was also observed with m708.2, whichhowever only partly blocked IGF-I induced phosphorylation at 10 nM. Inaddition to IGF-II, both m708.2, m708.5 and m610 were capable ofinhibiting IGF-II-induced phosphorylation of IGFIR (FIG. 6 b). However,m708.5 was superior to m708.2 and competed with m610. The antibodiesalso inhibited IGF-II-mediated phosphorylation of the IR induced by 5 nMof IGF-II (FIG. 7 a). The inhibition was independent on human insulinthat m708.5 did not bind (FIG. 7 b). Therefore, m708.5 could effectivelyinhibit ligand-induced phosphorylation of the two receptors withoutinhibiting insulin-IR interaction.

Example 8 Inhibition of MCF-7 Cell Growth

In order to investigate whether the blocking of IGF binding by m708.5 ispotent enough to inhibit the proliferation of cells, the activity ofm708.5 was tested in a cell growth assay using MCF-7 cells. MCF-7 cellsproduce significant amounts of IGF-II-concentrations up to 35 nM wereobserved after 3 days (Feng et al., Mol Cancer Ther, 2006. 5(1): p.114-20). After 3 days, cancer cells were inhibited by near 100% at 320nM of m708.5 (FIG. 7). Above 40 nM m708.5, the treatment still resultedin obvious cell growth inhibition. In contrast, m708.2 could onlyinhibit cancer cells beyond 80 nM. These data show that inhibitionability of m708.5 is remarkable, and that this antibody can effectivelyblock tumor cell proliferation in vitro.

The IGF signaling system plays an important role in tumorigenesisLeRoith and Roberts, Cancer Lett, 2003. 195(2): p. 127-37). Human IGF-Iand IGF-II share a 62% sequence homology and have overlapping functions:both IGF-I and IGF-II can activate the IGF-IR driving tumor cellproliferation. To further explore novel human antibodies against IGFsystem, three human antibodies specific for IGF-I utilizing phagedisplay technologies were identified. One of these antibodies, m708,exhibited significant binding to both IGF I and IGF II and was furtheraffinity matured by light-chain shuffling and mutagenesis to finallyselect a very high affinity antibody, m708.5. This antibody potentlyinhibited both IGF-1- and IGF-II-induced phosphorylation of IGF-IR, andthe growth of cancer cells expressing IGF-IR.

The inhibitory activity of the antibodies was cell type dependent with alikely major determinant the surface concentration of the IGF-IR and theinsulin receptor. Many IGF-IR-specific antibodies have been underpreclinical study, and several are being evaluated in clinical trials(Pollack et al., Nat Rev Cancer, 2008. 8(12): p. 915-28). For example, afully human monoclonal antibody (m610) with high affinity to IGF-II wasreported that potently blocked the growth/migration of human cancerlines in vitro (Feng et al., Mol Cancer Ther, 2006. 5(1): p. 114-20),and significantly suppressed the growth of prostate cancer cells in ahuman bone environment (Kimura et al., Clin Cancer Res, 2010. 16(1): p.121-9). A murine mAb cross-reactive to human IGF-I and IGF-II inhibitedthe development of new bone tumors, and the progression of establishedtumors (Goya et al., Cancer Res, 2004. 64(17): p. 6252-8). Recently, anew mAb against IGF-I and IGF-II was reported.

The antibody m708.5 should also exhibit inhibitory activity in vivo. Thefinding that IgG m708.5 has significantly higher affinity than scFv isimportant because it would allow to use the IgG antibody format, whichis most stable and has longest half-life in vivo; the IgG format mayalso confer certain effector functions in vivo.

Human monoclonal antibody m708.5 is cross-reactive to both IGF-I andIGF-II with picomolar affinity and potently inhibits the IGF-IR signaltransduction function. Thus, m708.5, and the antibodies disclosedherein, offer a new and promising therapeutic strategies for treatingtumor and a therapeutic alternatives to agents that target the IGF-IRitself.

Example 9 Bispecific Antibodies

A schematic representation of an exemplary of a bispecific antibody isshown in FIG. 11. To create m67, scFv m610.27 and scFv m708.5 were fusedto the N terminus of the heavy and light chain constant regions of ahuman IgG1, respectively, using linkers of G₄S triplicates.

The antibodies were then tested to determine the epitope of IGF-II boundby the antibody. For the results shown in FIG. 12A, IGF-II was directlycoated on the ELISA plate. Bound scFv m610 was detected by anti-Flagantibody-HRP in the presence of competing antibody (IgG1 m708.5 or IgG1m102.4.). M102.4 is an isotype control IgG1 that does not recognizeIGF-II. The result indicates that adding m708.5 to the antibody solutiondoes not diminish the m610 binding, indicating that m610 and m708.5 havedifferent epitopes on IGF-II. For the results shown in FIG. 12B, IgG1m610.27 was coated on the ELISA plate and IGF-II was captured by coatedm610.27. Bound scFv m708.5 or VH m630.3 was detected by HRP-conjugatedanti-Flag tag antibody. VH m630.3 is an anti-IGF-II antibody that isknown to have different epitopes from m610. FIG. 12B indicates thatm708.5 binds to IGF-II on a different epitope from m610.27.

The nature of the complex of m67 and IGF-II was then investigated. Forthe results shown in FIG. 13A, IgG1 m708.5 or the mixture of IgG1 m610and IgG1 m708.5 plus IGF-II was analyzed by Superdex G75 column. Becausem610 and m708.5 have different epitopes on IGF-II and each antibody isbivalent, together they form multimer complex, which are eluted from theSupedex75 column much early than the m708.5 alone. As shown in FIG. 13B,m67, the mixture of m67 and IGF-II, or the mixture of m67/IGF-II plusIGF-I was analyzed by Superdex G200 column. The bispecific antibody,m67, and IGF-II form similar large complex.

The stability of m67 and m708.5 in human sera was also evaluated. Asshown in FIG. 14, m67 (FIG. 14A) an dm708.5 (FIG. 14B) were incubatedwith equal volume of human sera at 37° C. for 9 days and then tested tobind to IGF-I and IGF-II by ELISA. The result indicated that bothantibodies retains their binding ability after incubation with serum for9 days.

m67 inhibited the binding of IGF-I (FIG. 15A) and IGF-II (FIG. 15B) toMCF7 cells better than m610.27 and m708.5 alone. For these studies,MCF-7 cells were incubated with 5 nM biotinylated IGF-I or 1 nMbiotinylated IGF-II in the absence or presence of antibodies. Boundbiotinylated IGF-I or IGF-II was detected by streptavidin-PE. Controlcells incubated with streptavidin-PE only were indicated with blackfills. m610.27 did not inhibit the binding of IGF-II to cells at theconcentration used in the assay due to its relatively low affinity.m708.5 inhibited the binding of IGF-I better than that of IGF-II.However, the bi-specific antibody m67 was able to inhibit both IGF-I andIGF-II from binding to cells. The result indicates that the two bindingmoieties of m67 have a synergistic effect.

The ligand/antibody complexes formed between IGF-II/m67 orIGF-II/m610.27+m708.5 are able to bind to Fc gamma receptor II on BJABcells (see FIG. 16). For these studies, bound biotinylated IGF-I orIGF-II was detected by Streptavidin-PE. BJAB cells are known to have lowaffinity Fc gamma receptor II, which only binds to a cluster of IgGmolecules but not to a single IgG. The mono-specific antibody, m610.27and m708.5 each recognized a single epitope on IGF-II, therefore, theydid not form multi-IgG complex with IGF-II. They also did not bind tothe BJAB cells. The bi-specific m67 forms multi-IgG complex with IGF-II.With multiple copies of IgG on the complex, the avidity effect renderedbinding ability to BJAB cells. The mixture of m610.27 and m708.5functions similarly to m67.

IGF-II/m67 complexes are endocytosed by macrophage-like U937 cells,whereas IGF-II/m610.27 or IGF-II/m708.5 complexes are not taken in byU937 cells (see FIG. 17). U937 cells have high affinity receptor forIgG. Both mono-specific and bi-specific antibodies are able to bind toU937 cells. However, only the m67/IGF-II complex is efficiently taken indue to the multiple copies of IgG on the complex. The two mono-specificantibodies, m610.27 and m708.5, do not form multimers with IGF-II andare not endocytosed by U937 cells. The endocytosis of m67/IGF-II complexis inhibited by cytochalasin D, an inhibitor of actin polymerization.The endocytosis process is dependent on the actin filaments.

m67 had stronger inhibition on IGF1R and IR phosphorylation than theparental mono-specific antibodies (see FIG. 18). Specifically, m67inhibited IGF-I induced phosphorylation of IGF-1R at 1 nM, similar tom708.5. It inhibited IGF-II induced phosphorylation of IGF-1R at 0.2 nM,better than both m610.27 and m708.5. Because m610.27 only recognizesIGF-II but not IGF-I, the ability of m67 to inhibit IGF-I bindingderives from m708.5 alone. However, because m610.27 and m708.5 bind totwo non-overlapping epitopes on IGF-II, m67 inhibited IGF-II bindingbetter than the single antibody, again providing evidence of asynergistic effect.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

The invention claimed is:
 1. An isolated human monoclonal antibody, orantigen binding fragment thereof, wherein the monoclonal antibody orantigen binding fragment comprises a heavy chain variable region and alight chain variable region, wherein the heavy chain variable regioncomprises the amino acid sequence set forth as amino acids 26-33 of SEQID NO: 7, amino acids 51-58 of SEQ ID NO: 7, and amino acids 97-109 ofSEQ ID NO: 7, wherein residue 56 is G and residue 109 is N; and whereinthe light chain variable region comprises the amino acid sequence setforth as amino acids 27-32 of SEQ ID NO: 8, amino acids 50-52 of SEQ IDNO: 8 and amino acids 89-97 of SEQ ID NO: 8, wherein the humanmonoclonal antibody or antigen binding fragment specifically bindsinsulin-like growth factor II (IGF-II) with an equilibrium dissociationconstant (K_(d)) of 200 pM or less and specifically binds IGF-I with anequilibrium dissociation constant (K_(d)) of 200 pM or less.
 2. Theisolated human monoclonal antibody or antigen binding fragment of claim1, wherein the heavy chain variable domain comprises the amino acidsequence set forth as SEQ ID NO: 7, wherein residue 7 is L; residue 20is V; residue 23 is K; residue 37 is V; residue 56 is G; residue 61 isA; residue 63 is Q; residue 74 is K; residue 76 is T; residue 77 is S;residue 79 is A; residue 85 is S; residue 87 is G; residue 109 is N;residue 111 is G; and residue 115 is L.
 3. The isolated human monoclonalantibody of claim 1, wherein the antibody is an IgG or IgM.
 4. Theisolated human monoclonal antibody of claim 3, wherein the antibody isan IgM or an IgG₄.
 5. The antigen binding fragment of claim 1, whereinthe antigen binding fragment is a Fab′ fragment, a F(ab)′2 fragment, asingle chain Fv protein (scFv), or a disulfide stabilized Fv protein(dsFv).
 6. An isolated bispecific antibody comprising a first monoclonalantibody, or antigen binding fragment thereof, and a second monoclonalantibody, or antigen binding fragment thereof, wherein the firstmonoclonal antibody comprises the human monoclonal antibody or antigenbinding fragment of claim 1, and the second monoclonal antibody orantigen binding fragment comprises a heavy chain variable regioncomprising amino acids 26-33 of SEQ ID NO: 23, amino acids 51-58 of SEQID NO: 23 and amino acids 97-109 of SEQ ID NO: 23, and a light chainvariable region comprising amino acids 27-32 of SEQ ID NO: 24, aminoacids 50-52 of SEQ ID NO: 24 and amino acids 89-98 of SEQ ID NO: 24,wherein the first monoclonal antibody or antigen binding fragmentthereof specifically binds IGF-I and IGF-II, and wherein the secondmonoclonal antibody or antigen binding fragment thereof specificallybinds IGF-II.
 7. The isolated human monoclonal antibody or antigenbinding fragment of claim 1, wherein the monoclonal antibody or antigenbinding fragment is labeled.
 8. The isolated human monoclonal antibodyor antigen binding fragment of claim 7, wherein the label is afluorescence, enzymatic, or radioactive label.
 9. The isolated humanmonoclonal antibody or antigen binding fragment of claim 1, wherein theantibody or antigen binding fragment binds IGF-I with an equilibriumconstant (K_(d)) of 200 pM or less and binds IGF-II with an equilibriumconstant (K_(d)) of 60 pM or less.
 10. A composition comprising thehuman monoclonal antibody or antigen binding fragment of claim 1, and apharmaceutically acceptable carrier.
 11. A method of detectinginsulin-like growth factor (IGF)-I and/or IGF-II in a biological samplefrom a subject, comprising contacting the biological sample from thesubject with the composition of claim 10; and detecting binding of thehuman monoclonal antibody or antigen binding fragment to the biologicalsample, wherein an increase in the binding of the human monoclonalantibody or antigen binding fragment to the biological sample ascompared to a control indicates that IGF-I and/or IGF-II is present inthe sample.
 12. The method of claim 11, wherein the human monoclonalantibody or antigen binding fragment is directly labeled.
 13. The methodof claim 11 wherein the biological sample is assayed by competitionimmunoassay.
 14. The method of claim 11 wherein the sample is tissuefrom a biopsy, autopsy, or pathology specimen.
 15. The method of claim11, wherein the sample is a blood, urine, biopsy, serum, sputum, plasma,cerebral spinal fluid sample.
 16. A method of inhibiting phosphorylationof the insulin-like growth factor-I receptor, comprising contacting acell that expresses IGF-I receptor on its cell surface with an effectiveamount of the composition of claim 10, thereby inhibiting thephosphorylation of the insulin-like growth factor receptor.
 17. Themethod of claim 16, wherein the cell is in vitro.
 18. The method ofclaim 16, wherein the cell is in vivo.
 19. The method of claim 16,wherein the cell is a cancer cell.
 20. A method of treating a subjectwith breast cancer, comprising administering to the subject atherapeutically effective amount of a composition comprising theisolated monoclonal antibody or antigen binding fragment of claim 1 toreduce tumor burden, tumor metastasis, or both, thereby treating thebreast cancer in the subject.
 21. The method of claim 20, whereinadministering the therapeutically effective amount of the compositionreduces breast cancer metastasis.
 22. The method of claim 20, whereinadministering the therapeutically effective amount of the compositioninhibits the phosphorylation of the IGF-I receptor.
 23. The isolatedhuman monoclonal antibody of claim 1, wherein the monoclonal humanantibody inhibits the motility of breast cancer cells in vitro.
 24. Theisolated human monoclonal antibody of claim 1, wherein the antibodyinhibits phosphorylation of the insulin-like growth factor receptor. 25.An isolated bispecific antibody comprising a first scFv that binds IGF-Iand IGF-II and a second scFv that binds IGF-II, wherein: the first scFVcomprises a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO: 7, wherein residue 7 is L; residue 20 is V;residue 23 is K; residue 37 is V; residue 56 is G; residue 61 is A;residue 63 is Q; residue 74 is K; residue 76 is T; residue 77 is S;residue 79 is A; residue 85 is S; residue 87 is G; residue 109 is N;residue 111 is G; and residue 115 is L, and a light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 8, whereinresidue 4 is M; residue 22 is V; residue 33 is V; residue 53 is S;residue 58 is V; residue 72 is A; and residue 108 is Q; and the secondscFv comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 23, and a light chain variable domain comprisingthe amino acid sequence of SEQ ID NO:
 24. 26. The bispecific antibody ofclaim 25, wherein the first scFv and second scFv are linked usinglinkers of G₄S triplicates.
 27. The bispecific antibody of claim 25,wherein the first scFv is fused to the N-terminus of the light chainconstant region of an IgG1 and the second scFV is fused to theN-terminus of the heavy chain constant region of the IgG1, and whereinthe first scFv and second scFv are fused to the IgG1 light chain andheavy chain constant regions using linkers of G₄S.